ENRICHED PLANT EXTRACT, COMPOUND, AND METHODS FOR THE TREATMENT OF PROLIFERATIVE DISORDERS

FIELD OF THE INVENTION The present invention relates to an enriched plant extract effective for the treatment of proliferative disorders such as cancer. More particularly, the present invention relates to an enriched plant extract including a mixture of two compounds present in the percentage ratio of about 10:90 to about 90:10, obtained from the leaves of the plant Vitex negundo. The mixture exhibits anti-proliferative effects on several cancer cell-lines. The invention also relates to a compound of formula 1 . The compound of formula 1 can be isolated from the mixture of two compounds. This compound has anti-proliferative activity.

The present invention further relates to a herbal composition including enriched extract of leaves of Vitex negundo containing the mixture of two compounds as main ingredient and method of manufacture of the same. The invention also relates to a pharmaceutical composition including the compound of formula 1 , and method of manufacture of the same. The invention further relates to the method of treatment of proliferative disorders by administering the compositions to a mammal in need thereof.

BACKGROUND OF THE INVENTION

In cancer, the balance between proliferation and programmed cell death is disturbed, and certain genetic disturbances of apoptotic signaling pathways have been found in carcinomas leading to tumor development and progression. Type Il topoisomerases (topoisomerase Il alpha and topoisomerase Il beta) are crucial enzymes required during DNA replication and cell division and therefore are ideal targets for anticancer therapy. Topoisomerase Il alpha is minimally expressed in normal cells. In contrast, over expression of topoisomerase Il alpha has been found in breast cancer and other major cancer types. Extrinsic and intrinsic pathways are the two major signaling pathways of apoptosis that involve death receptors and mitochondria. Dysregulation of one or both apoptotic pathways is common in many types of cancers and can occur due to inactivation of apoptosis promoting proteins (p53 and Bax) and/or hyperactivity of

survival or anti-apoptotic genes (for example: Bcl-2). One of the most commonly detected abnormalities in human cancer is mutation of the p53 tumor suppressor gene.

The mitochondrial pathway is thought to be important in response to cancer treatment and is mediated by Bcl-2 family proteins. In addition, certain nuclear transcription factors like cjun and JNK (N-terminal c-jun kinase) have also been implicated in the signal cascade of events that lead to apoptosis. The final execution of cell death is performed by the caspase cascade, which is triggered by release of cytochrome C (mitochondrial protein) from mitochondria as reported in Anticancer Agents Med. Chem., 2006, 6(4), 303-18.

The genus Vitex of the family Verbenaceae is constituted by 250 species of small trees and shrubs which occur in tropical to temperate regions. Vitex negundo is a small aromatic plant that is widely distributed in South Asia, the People's Republic of China, Indonesia, and the Philippines. The fresh leaves of the plant have anti- inflammatory, analgesic and antihistamine activities among a variety of other activities as reported in Journal of Ethnopharmacology, 2003, Vol.87, 199 - 206 and Wealth of India, Vol. X, 522 - 524. The extract of the leaves exhibits anti-cancer activity due to a constituent of the leaves being a cytotoxic flavone, vitexicarpin as reported in J. Nat. Prod., 2003, Vol.66, 865 - 867.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides an enriched extract containing a mixture of two compounds, obtained from leaves of Vitex negundo, which is effective for inhibiting proliferation of cancer cells or treating cancer. Another aspect of the present invention provides the compound of formula 1 (as described herein below) having antiproliferative activity. The compound of formula 1 can be isolated from the enriched extract.

According to another aspect of the invention, the invention provides a method for reducing the proliferation of cancer cells, the method including contacting the cells with the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo.

A further aspect of the present invention, is a herbal composition including an enriched extract of the leaves of Vitex negundo containing the mixture of two compounds as main ingredient.

Further, the present invention provides a pharmaceutical composition for treating cancer, which includes a therapeutically effective amount of the compound of formula 1 along with a pharmaceutical carrier. The invention also includes methods of manufacture of the enriched extract containing the mixture of two compounds, of the compound of formula 1 , and of the compositions. According to a further aspect, the present invention provides a method for treating proliferative disorders such as cancer, which includes administering to a subject in need thereof, a therapeutically effective amount of the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo or the compositions thereof. Further scope of applicability of the present invention will become apparent from the detailed description to follow. It should be understood, however, that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : lntemucleosomal DNA Fragmentation Analysis of HEp-2 cells Figure 2:

A: RT-PCR analysis of p53 expression in HEp-2 cells

B: RT-PCR analysis of p53 expression in MDA-MB231 cells Figure 3:

A: RT-PCR analysis of Bcl-2 expression in HEp-2 cells

B: RT-PCR analysis of Bcl-2 expression in MDA-MB231 cells

Figure 4:

A: RT-PCR analysis of Bax expression in HEp-2 cells B: RT-PCR analysis of Bax expression in MDA-MB231 cells

Figure 5:

A: RT-PCR analysis of c-jun expression in HEp-2 cells B: RT-PCR analysis of c-jun expression in MDA-MB231 cells Figure 6: A: Western blot analysis of p53 protein expression in HEp-2 cells

B: Western blot analysis of p53 protein expression in MDA-MB231 cells Figure 7:

A: Western blot analysis of Bcl-2 protein expression in HEp-2 cells B: Western blot analysis of Bcl-2 protein expression in MDA-MB231 cells Figure 8:

A: Western blot analysis of Cytochrome C release in HEp-2 cells

B: Western blot analysis of Cytochrome C release in MDA-MB231 cells

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

The present invention relates to an enriched plant extract of leaves of Vitex negundo containing a mixture of two compounds present in the percentage ratio of about 10:90 to about 90:10. The mixture exhibits anti-proliferative effects on cancer cell- lines. The invention further relates to a compound of formula 1 .

Formula 1

In one aspect, the compound of formula 1 can be isolated from the above mentioned mixture of two compounds. The compound of formula 1 has anti-proliferative activity.

As used herein, the term "compound of formula 1 " includes a stereoisomer, a geometrical isomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.

The invention also relates to a herbal composition including an enriched extract containing the mixture of two compounds present in the percentage ratio of about 10:90 to about 90:10, obtained from the leaves of Vitex negundo and method of manufacture of the same. The invention further relates to a pharmaceutical composition including the compound of formula 1 and a pharmaceutical carrier and method of manufacture of the same. These compositions can be employed for the treatment of proliferative disorders like cancer.

The plant used in this study was collected from the Western Ghats near Kotagiri in the Nilgiris district, Tamil Nadu, India. A microscopic and macroscopic study for authentication of Vitex negundo was carried out. A specimen is retained in Botany Department, Nicholas Piramal Research Centre, Goregaon, Mumbai, Maharashtra, India.

" Vitex negundo extract", as used herein means a blend of compounds present in the plant Vitex negundo. Such compounds can be extracted from the dried leaves of the plant using extraction procedures well known in the art (e.g., the use of organic solvents such as lower alcohols, alkyl esters, alkyl ethers, alkyl ketones, chloroform, petroleum ether, hexane and/or inorganic solvents such as water). The plant material can be extracted using ethyl acetate.

The present process for preparation of the extract of the leaves of Vitex negundo can be scaled up for large-scale preparation. Vitex negundo crude extract can be standardized using conventional techniques such as high performance liquid chromatography (HPLC). The term "standardized extract" refers to an extract which is standardized by identifying and quantifying characteristic bioactive ingredient(s) present in the extract.

The preliminary activity determination of the extracts can be carried out using various well-known biological assays. Bioactive ingredients can be identified using various techniques such as thin layer chromatography (TLC) or HPLC. Bioactive ingredients can be isolated from the extract of leaves of Vitex negundo by bioactivity guided column chromatographic purification and preparative high performance liquid chromatography (HPLC).

In a further embodiment, the enriched Vitex negundo extract contains a mixture of two compounds in the percentage ratio of about 50:50, which can be measured using conventional assay techniques such as high performance thin layer chromatography (HPTLC) or high performance liquid chromatography (HPLC). In a yet further embodiment, the enriched Vitex negundo extract contains a mixture of two compounds in the percentage ratio of about 90:10, which can be measured using conventional assay techniques such as high performance thin layer chromatography (HPTLC) or high performance liquid chromatography (HPLC). In yet another embodiment, the invention relates to a method for isolating and characterizing the compound of formula 1 from an enriched extract containing the mixture of two compounds in a percentage ratio of about 90:10. The enriched extract containing the mixture of two compounds was obtained by column chromatography followed by preparative HPLC. After evaluating the LC-MS & MS-MS spectra of the mixture of two compounds, it was observed that both the compounds have a molecular weight of 472.

The enriched extract containing the mixture of two compounds was further subjected to preparative HPLC to obtain the pure compound labeled as formula 1 . The structure of the compound was assigned by interpretation of its spectral data to be as follows;

Formula 1

It will be appreciated by those skilled in the art that the compound of the present invention can also be utilized in the form of its pharmaceutically acceptable salts or solvates thereof. The pharmaceutically acceptable salts of the compound of the present invention are in particular salts which are non-toxic, or which can be used physiologically.

The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds which are prepared with acids or bases, depending on the particular substituents found on the compounds described herein. Since compound of formula

1 contains acidic functionality, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.

The neutral forms of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

The compound of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. The compound of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Those skilled in the art will recognize that stereocentres exist in the compound of formula 1. Accordingly, the present invention includes all possible stereoisomers and geometric isomers of formula 1 and includes not only racemic compounds but also the optically active isomers as well. When a compound of formula 1 is desired as a single enantiomer, it may be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or any convenient intermediate. Resolution of the final product, an intermediate or a starting material may be effected by any suitable method known in the art for example Tables of Resolving Agents by S. H. Wilen. The present invention includes all possible geometric or cis-trans (E/Z) isomers of the compound of formula 1 . The invention includes both the E form and the Z form of compound of formula 1 , as well as mixtures of these forms in all ratios. The preparation of individual isomers can be carried out, if desired, by separation of a mixture by customary methods. The term "treating", "treat" or "treatment" as used herein includes preventive (prophylactic) and palliative treatment.

As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert, solid, semi-solid, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; malt; gelatin; talc; as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents; preservatives and antioxidants can also be present in the composition, according to the judgement of the formulator. As used herein, "safe and effective amount" means an amount of compound or composition (e.g., the Vitex negundo enriched extract or another composition including the compound of formula 1 , sufficient to significantly induce a positive modification in the condition to be regulated or treated, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgement. The safe and effective amount of the compound or composition will vary with the particular condition being treated, the age and physical condition of the end user, the severity of the condition being treated/prevented, the duration of the treatment, the nature of concurrent therapy, the specific compound or composition employed, the particular pharmaceutically acceptable carrier utilized, and like factors. As used herein, all percentages are by weight unless otherwise specified. As used herein, the term 'about' means an admissible range of ±10 %. For example, 'about 90 %' means 90 ± 10 %. Similarly 'about 50 %' means 50 ± 10 %. As used herein, 'bioactive ingredient' means the compound of formula 1 or the enriched extract containing the mixture of two compounds. In one embodiment, the bioactive ingredient(s) of the present invention are used in regulating cell proliferation. The bioactive ingredient(s) of the present invention are capable of inhibiting cell proliferation and are therefore useful in treating diseases, which are due to an excessive or abnormal cell growth. The bioactive ingredient(s) of the present invention are inhibitors of topoisomerase II. Inhibition of topoisomerase Il

causes downregulation of the function of monocytes and therefore, inhibitors of topoisomerase Il would be useful in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (Rheumatology, 2005, 44, 2, 183-186). There are a wide variety of pathological conditions with excessive or abnormal cell proliferation against which the compounds of the invention can act to provide therapeutic benefits. Examples of such pathological conditions include: a) various cancers and leukemias including the following: i. carcinoma, including that of bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin; ii. hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma, and Burkett's lymphoma; iii. hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; iv. tumors of mesenchymal origin, including fibrosarcoma and rhabdomysarcoma; and v. other tumors including melanoma, seminoma, teratocarcinoma, osteosarcoma, neuroblastoma and glioma, and b) inflammation and arthritis.

The present invention furthermore relates to pharmaceutical or herbal compositions that contain an effective amount of the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo respectively, in addition to a pharmaceutically acceptable carrier. The invention also includes a process for the production of the pharmaceutical or herbal composition, which includes bringing the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo respectively, into a suitable administration form using a pharmaceutically suitable and physiologically tolerable excipient.

In an embodiment, the compound of formula 1 or the enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, or the compositions thereof, exhibit in vitro anti-proliferative activity on cancer cells by inducing the apoptotic machinery and/or by stabilization of topoisomerase ll-alpha - DNA cross-link leading to the absence of free topoisomerase Il for subsequent DNA replication.

The invention also provides a method for reducing the proliferation of cancer cells. The method includes contacting the cells with the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, or the compositions thereof. An aspect of this invention is a method for treating a mammal (e.g., a human) suffering from cancer, by administering a therapeutically effective amount of the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, or the compositions thereof.

In an aspect of the invention, the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, or the compositions thereof, can be administered in an amount therapeutically effective to reduce the proliferation of cancer cells.

Another aspect of this invention is directed to a method for preventing and/or minimizing damage resulting from cancer, by administering to an affected mammal, (e.g., a female or male human), a therapeutically effective amount of the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, or the compositions thereof.

The present invention also relates to the use of the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, for the manufacture of a medicament for the prevention or treatment of cancer.

In an embodiment, the present invention relates to a method for the preparation of a medicament for the treatment or prevention of cancer comprising, the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, as pharmaceutically active substance.

The present invention further relates to a method for the preparation of a medicament including the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of Vitex negundo, for reducing the proliferation of cancer cells. The present invention accordingly relates to the use of the compound of formula 1 or enriched extract containing the mixture of two compounds, obtained from leaves of

Vitex negundo, for the manufacture of a medicament for the prevention or treatment of cancer.

The invention is further directed to a method of manufacturing a herbal composition useful for treating proliferative disorders. The enriched extract of Vitex negundo is mixed with a pharmaceutically acceptable carrier and formulated into therapeutic dosage forms.

The enriched extract of leaves of Vitex negundo is used to prepare oral or topical preparations containing about 5 to about 90 % by weight of the enriched extract, which is thoroughly blended into a conventional base. The plant extract contains about 10 to about 100 % (w/w) of the bioactive ingredient, which is sufficient to achieve the desired results.

In a further aspect, the invention is directed to a method of manufacturing a pharmaceutical composition for treating proliferative disorders. The compound of formula 1 is mixed with a pharmaceutically acceptable carrier and formulated into therapeutic dosage forms.

The pharmaceutical compositions normally contain about 1 to 99 %, for example, about 5 to 70 %, or about 10 to about 30 % by weight of the compound of formula 1 . The amount of the compound of formula 1 , and/or its physiologically tolerable salt and/or its isomer in the pharmaceutical compositions can, for example, be from about 5 to 500 mg. The dose of the compounds of this invention, which is to be administered, can cover a wide range. The dose to be administered daily is to be selected to suit the desired effect. A dosage of about 0.001 to 100 mg/kg/day of the compound of general formula 1 may be administered per day. If required, higher or lower daily doses can also be administered. In an aspect of the invention, the treatment methods and methods for reducing cellular proliferation described herein include the administration of herbal or pharmaceutical compositions described above, by known administration routes, modes, etc. including the following.

The pharmaceutical compositions according to the present invention can be administered orally, for example in the form of pills, tablets, coated tablets, capsules, granules or elixirs. Administration, however, can also be carried out rectally, for example in the form of suppositories, or parenterally, for example intravenously, intramuscularly or subcutaneously, in the form of injectable sterile solutions or

suspensions, or topically, for example in the form of solutions or transdermal patches, or in other ways, for example in the form of aerosols or nasal sprays. Actual dosage levels of the bioactive ingredients in the compositions of this invention can be varied so as to obtain an amount of the bioactive ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular extract of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular composition being employed, the duration of the treatment, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

In another embodiment, the present invention relates to reducing proliferation of cancer cells such as laryngeal carcinoma cell line (HEp-2 cells), breast carcinoma cell line (MDA-MB-231 cells), renal carcinoma cell line (ACHN cells), pancreatic carcinoma cell line (Panc-1 ), lung carcinoma cell line (Calu-1 ), human non-small-cell lung carcinoma (H-460), human colon carcinoma (HCT-1 16), human breast carcinoma (ZR-75) and human ovarian adenocarcinoma (A-2780), by contacting the cells with the enriched extract of Vitex negundo containing the mixture of two compounds in the percentage ratio of about 10:90 to about 90:10.

In another embodiment, the present invention relates to reducing proliferation of cancer cells such as laryngeal carcinoma cell line (HEp-2 cells), breast carcinoma cell line (MDA-MB-231 cells), renal carcinoma cell line (ACHN cells), pancreatic carcinoma cell line (Panc-1 ), lung carcinoma cell line (Calu-1 ), human non-small-cell lung carcinoma (H-460), human colon carcinoma (HCT-1 16), human breast carcinoma (ZR-75) and human ovarian adenocarcinoma (A-2780) by contacting the cancer cells with the compound of formula 1 .

Yet another embodiment of the present invention also relates to reducing proliferation of cancer cells such as laryngeal carcinoma cell line (HEp-2 cells), breast carcinoma cell line (MDA-MB-231 cells), renal carcinoma cell line (ACHN cells), pancreatic carcinoma cell line (Panc-1 ), lung carcinoma cell line (Calu-1 ), human non-small-cell lung carcinoma (H-460), human colon carcinoma (HCT-1 16), human breast carcinoma (ZR-75) and human ovarian adenocarcinoma (A-2780) by

contacting the cancer cells with the composition including the compound of formula 1 or the enriched extract containing the mixture of two compounds in the percentage ratio of about 10:90 to about 90:10.

The enriched extract containing the mixture of two compounds in the percentage ratio of about 10:90 to about 90:10, of the present invention is suitable for use in the treatment of acute or chronic forms of cell proliferation disorders such as cancer. The present invention includes administering to a subject in need thereof the enriched extract for treating acute or chronic forms of cell proliferation disorders such as cancer. In an embodiment, the said enriched extract is used in the treatment of breast, colon, kidney, pancreas, lung, non-small-cell lung, ovarian or laryngeal carcinoma. The compound of formula 1 of the present invention is suitable for use in the treatment of both acute and chronic forms of cell proliferation disorders such as cancer. The present invention includes administering to a subject in need thereof the compound of formula 1 for treating acute or chronic forms of cell proliferation disorders such as cancer. In an embodiment, the said compound is used in the treatment of breast, colon, kidney, pancreas, lung, non-small-cell lung, ovarian or laryngeal carcinoma. The compositions of the present invention are suitable for use in the treatment of both acute and chronic forms of cell proliferation disorders such as cancer. The present invention includes administering to a subject in need thereof the present compositions for treating acute or chronic forms of cell proliferation disorders such as cancer. In an embodiment, the said compositions are used in the treatment of breast, colon, kidney, pancreas, lung, non-small-cell lung, ovarian and laryngeal carcinoma. The bioactivity guided purification and isolation of enriched bioactive ingredient was achieved as described in detail in subsequent examples. The efficacy of the present enriched plant extract has been established by biological assays well known in the art and are described in detail in subsequent examples. The following examples illustrate but do not limit the scope of the invention. It is to be understood by those of the ordinary skill in the art that the present discussion is of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary construction.

The following terms/abbreviations/chemical formulae are employed in the Examples:

ATCC : American Type Culture Collection

1 X Tris-borate-EDTA : Tris-borate-Ethylene Diamine Tetra Acetic acid buffer

³ [H]-dT : Tritiated Thymidine

BCIP : 5-Bromo-4-Chloro-3-lndolylphosphate p-Toluidine Salt

BSA : Bovine Serum Albumin

CDNA : Complementary DNA

CPM/mg protein : Count Per Minute/milli gram Protein

CsCI : Cesium chloride

CO ₂ : Carbon dioxide

DEPC : Diethyl Pyrocarbonate

DMSO : Dimethyl Sulfoxide

DNA : Deoxyribonucleic acid dNTP : Deoxyribonucleotide triphosphate

EDTA : Ethylene Diamine Tetra Acetic acid

FACS : Fluorescence Activated Cell Sorter

GAPDH : Glyceraldehyde Phosphate Dehydrogenase

GuHCI : Guanidine hydrochloride

HBSS : Hank's Balanced Salt Solution

HCI : Hydrochloric acid

IPA : lsopropyl alcohol

KCI : Potassium chloride

MgCI ₂ : Magnesium chloride

NaCI : Sodium chloride

NaOH : Sodium hydroxide

NBT : p-Nitro-Blue Tetrazolium Chloride

NBT-BCIP : p-Nitro-Blue Tetrazolium Chloride - 5 - bromo - 4 chloro - 3 -indolylphosphate p-toluidine salt

PBS : Phosphate Buffered Saline

PCR : Polymerase Chain Reaction

RNA : Ribonucleic acid

SDS : Sodium dodecyl sulphate

Taq : Thermophilus aquaticus

TCA : Trichloroacetic acid TE buffer : Tris Ethylene Diamine Tetra Acetic acid

Example 1 : Preparation of crude extract.

Example 1A : Shade dried leaves (500 g) of Vitex negundo were pulverized to 60- 120 mesh. The powdered material was extracted using ethyl acetate (1.0 L) with continuous stirring at 37 ⁰ C for 18 hours. The extract was then filtered and the residue was re-extracted twice. All extracts were then pooled and concentrated to dryness at 50 ⁰ C under reduced pressure. The weight of the obtained extract was 50

9-

Example 1 B : Shade dried leaves (500 g) of Vitex negundo were pulverized. The powdered material was extracted using ethyl acetate (1 .0 L) with continuous stirring at 50 ⁰ C for 18 hours. The extract was then filtered and the residue was re-extracted twice. All extracts were then pooled and concentrated to dryness at 50 °C under reduced pressure. The weight of the obtained extract was 50 g.

Example 2:

Preparation of enriched extract. Example 2A: 5 g of crude extract, obtained from Example 1 A was purified by column chromatography (silica gel; 60 - 120 mesh; hexane-ethyl acetate). Based on the TLC profile, fractions were pooled and tested for bioactivity. All fractions were tested at 50 μg/mL concentration. The activity of the different fractions is as indicated in Table 1 below. Table 1 : Anti-proliferative effect of column fractions on Hep-2 cell line.

Fraction F5 which showed the maximum anti-proliferative activity was further purified by column chromatography (silica gel; 100 - 200 mesh; hexane-ethyl acetate). The fractions were pooled on the basis of TLC and concentrated. Yield: 180 mg. The HPLC indicated that this enriched extract was a mixture of two compounds in the percentage ratio of 55:45. The analytical HPLC conditions used were as incorporated in Example 3.

Example 2B : 50 g of crude extract, obtained from Example 1 B was purified by column chromatography (silica gel; dichloromethane - isopropanol). The collected fractions were individually concentrated under reduced pressure at 50 ⁰ C. The evaporated fractions were re-constituted in minimum volume of dichloromethane (25 mL) and evaluated by TLC (silica gel; dichloromethane : isopropanol (90 : 10);1 % ammonium molybdate reagent). The fractions were pooled on the basis of TLC and concentrated to give the enriched extract obtained in a yield of 4.4 g, which was evaluated by HPLC. The HPLC indicated that this enriched extract was a mixture of two compounds in the percentage ratio of 40:60. The analytical HPLC conditions used were as incorporated in Example 3.

Example 3: Preparation of an enriched extract containing mixture of two compounds.

The enriched extract of Example 2B was further purified by gradient preparative

HPLC using the following conditions:

Column : Eurosphere RP Ci ₈ column (250 x 16 mm), 10 μ; Mobile phase : Acetonitrile : Water

Run time : 30 minutes

Flow rate : 20 mL/minute

Detection : UV 210 nm

Stock solution used : 100 mg / mL in Methanol Injection volume : 1 mL per injection

Total number of runs : 30

Table 2:

Details of the gradient used:

Three fractions were collected between 12.0 - 17.5 minutes.

1 ^st Fraction - Fraction collected between 12.0 minutes - 13.5 minutes referred to as Example 3-Fraction A obtained in a yield of 85 mg

2 ^nd Fraction -Fraction collected between 13.5 minutes - 15.8 minutes referred to as Example 3-Fraction B obtained in a yield of 105 mg

3 ^rd Fraction - Fraction collected between 15.8 minutes - 17.5 minutes referred to as

Example 3-Fraction C obtained in a yield of 56 mg

All three fractions were concentrated and freeze-dried. The dried fractions were then dissolved in methanol and re-injected to evaluate the HPLC profile using the gradient analytical method as indicated below.

Column : Eurosphere RP C18 column (250 x 4.6 mm), 5 μ Mobile phase : Acetonitrile : Water Run time : 40 min Flow rate : 1 mL/min Detection : UV 210 nm Stock solution used : 0.1 mg / ml_ in Methanol Injection volume : 50 μl_ per injection

Table 3:

Details of the gradient used:

The chromatograms showed presence of two peaks indicative of presence of mixture of two compounds in all the three fractions. The retention times of the two compounds in the above HPLC system were 17.5 and 18.2 minutes respectively which matched the retention times of the two compounds present in the enriched extract of Example 2A as well as Example 2B.

Example 3-Fraction A contained the two compounds in the percentage ratio of 89:1 1 ; Example 3-Fraction B contained the two compounds in the percentage ratio of 40:60 and

Example 3-Fraction C contained the two compounds in the percentage ratio of

27:73.

After evaluating the LC-MS & MS-MS spectra of the mixture of two compounds, it was observed that both the compounds have a molecular weight of 472.

Example 4: Isolation of pure compound of formula 1.

The enriched extract containing mixture of two compounds referred to as Example 3- Fraction A was further purified by gradient preparative HPLC employing the following conditions:

Column Eurosphere RP Ci ₈ column (250 x 16 mm), 10 μ

Mobile phase Acetonitrile : Water

Run time 30 minutes

Flow rate 20 mL/minute

Detection UV 210 nm

Stock solution used 7 mg/mL

Injection volume 1 mL per injection

Total number of runs : 1 1

Table 4:

Details of the gradient used:

Two fractions were collected per run between 12.0 - 14.9 minutes.

1 ^st fraction: 1 ^st fraction collected from the start of the 1 ^st peak to the peak height of 1 ^st peak.

2 ^nd fraction: 2 ^nd fraction collected from peak height of 1 ^st peak to the end of the 1 ^st peak.

The HPLC pure fractions obtained from the above runs were pooled together and concentrated to yield 5.4 mg pure compound of formula 1 , having a retention time of

1 1 minutes. The analytical HPLC conditions (gradient) are as indicated below.

Column : Kromasil C18 (15O x 4.6 mm), 3.5 μ

Mobile phase : Acetonitrile : Water

Run time : 30 minutes Flow rate : 1 mL/min

Detection : UV 210 nm

Stock solution used : 0.1 mg/mL in Methanol

Injection volume : 20 μL per injection

Temperature : 30 ⁰ C Table 5:

Details of the gradient used:

The compound of formula 1 was confirmed by spectral analysis to have the following structure:

Formula 1 The physico-chemical and spectral properties of the compound of formula 1 :

1. Appearance: White amorphous powder 15 2. Solubility: Ethyl acetate, methanol and DMSO

3. HPLC: As described in Example 4

4. Molecular formula of the compound: C _3I H ₅₂ O ₃

5. Molecular weight of the compound: 472.0

6. Mass spectral data (with fragmentation pattern): m/z (EI-MS) 472 [M]+, 454 0 (18), 436 (12), 426 (32), 408 (30), 393 (17), 300 (1 1 ), 248 (98), 203 (53), 189 (6),

175 (3), 133 (6), 109 (1 ), 95 (1 ).

7. Ultra-violet (UV) spectral data: 204 nm (from HPLC elution profile) 5 8. Fourier Transform-Infrared (FT-IR) spectral data (KBr; cm ^'1 ):

3423.28, 2944.20, 1699.52, 1508.13, 1464.70, 1387.77, 1378.16, 1366.29, 1304.0, 1263.77, 1210.14, 1 185.52, 1 162.64, 1096.20, 1050.51 , 1031.27, 993.26, 864.29, 822.87, 801 .88, 758.13, 659.34, 607.84, 561 .96

30 9. ¹ H NMR and ¹³ C NMR: Refer to Table 6

35

0

Table 6 : ¹ H-NMR spectral (DMSO-d ₆ , 500 MHz) data and ¹³ C-NMR spectral (DMSOd ₆ , 125 MHz) data:

Biological assays In vitro assays

The HEp-2 and M D A- M B -231 cell lines used in examples 5-12 were obtained from

ATCC (USA).

Example 5: [ ³ H]- Thymidine incorporation studies

The assay was designed as in reference, British Journal of Cancer, 2002, Vol.87, 98- 105, the disclosure of which is incorporated by reference for the teaching of the assay.

[ ³ H] -Thymidine (1 μCi/ ml_) was added to the cell before 24 hours from harvesting of

K) treated cells. Extracts were checked for antiproliferative activity in a 24 well plate with different concentration [from 100 to 1 μg, each dissolved in 4 μl_ of DMSO so that the final concentration in DMSO was 0.4 %]. Each well contains 1 x 10 ⁶ cells in 1 ml_ of medium. 4 μl_ of DMSO was used as solvent control. The plate was incubated for 12, 24 and 36 hours, then the cells were trypsinized at the desired time

15 points, pelleted and washed sequentially with 10 % and 5 % TCA and solubilised in a solution containing 0.1 N NaOH and 0.0025 % SDS. The radioactivity of the samples was measured in the WALLAC 1409 Liquid scintillation counter and protein was estimated by Lowry's method and the data was expressed as CPM/mg protein. Anti-proliferative effect of crude extract of Example 1 A and enriched extract of 0 Example 2A was studied in HEp-2 cells by [ ³ H] thymidine incorporation assay. 1 μg/mL to 100 μg/mL of sample was treated for 12, 24 and 36 hours. The results are depicted in Table 7. Table 7 : Dose response analysis in HEp-2 Cells

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Conclusion: The results indicated that the crude extract of Example 1 A and the enriched extract of Example 2A exhibited dose dependant anti-proliferative activity.

Example 6: Fluorescence Activated Cell Sorting (FACS) Analysis HEp-2 cells were grown in a 6 well plate and 10 μg of crude ethyl acetate extract of Example 1 A and enriched extract of Example 2A were added and incubated for 24 hours. The cells were then trypsinised and collected in micro centrifuge tubes and were resuspended in 50 μl_ of PBS or HBSS. Cells were fixed with ice-cold 70 % ethanol at -20 ⁰ C overnight (18 hours). 5 μl_ of Ribonuclease (1 mg/mL) was added to each tube and 50 μl_ of Propidium Iodide (25 μg/mL in PBS or HBSS) was added and incubated at 4 ⁰ C for 18 hours. The cells were then analysed by FAC Star PLUS (Becton Dickinson).

Percentage of apoptosis was analysed against propidium iodide (Pl) fluorescence by Flow cytometry. The extent of apoptosis was higher in treated samples than in untreated cells. Crude extract of Example 1 A and enriched extract of Example 2A showed 58.93 % and 51 .64 % respectively (control showed 5.49 %).

Example 7: DNA fragmentation assay a) TKM- 1 buffer (1 OmM Tris HCI-pH7.6, 1 OmM MgCI ₂ , 1 OmM KCI, 2mM EDTA), TKM-2 buffer (1 OmM Tris HCI- pH 7.6, 1 OmM MgCI ₂ , 1 OmM KCI, 0.4M NaCI, 2mM

EDTA), 5M NaCI, 10 % SDS were prepared. b) Low-molecular weight DNA was extracted from apoptotic cells. Cells were suspended in HBSS and incubated with 70 % ethanol at -20 ⁰ C for 24 hours. The resulting cells were centrifuged to remove ethanol, and the cell pellets were re- suspended and incubated in 200 μL of TKM-1 buffer and kept at the room temperature for 20 minutes. After centrifugation at 10,000 rpm for 10 minutes, 200 μL of TKM-2 buffer and 20 μL of 10 % SDS was added to the pellet. After incubation of 5 minutes, 2 μL of Ribonuclease [10 mg/mL] was added and incubated at 55 ⁰ C for 10 minutes. 125 μL of 5M NaCI was added and mixed well. After centrifugation at 10,000 rpm for 5 minutes, 2 volumes of ethanol was added to the supematent and kept at - 80 ⁰ C for 30 minutes. The pellet obtained after centrifuging at 12,000 rpm for 10 minutes, was washed with 70 % ethanol, centrifuged at 10,000 rpm and dried at 37 ⁰ C. The pellet was later dissolved in 20 μL of TE buffer (pH 8.3). The isolated

DNA was analyzed by 2 % agarose gel electrophoresis in 1 X Tris - borate - EDTA buffer with ethidium bromide staining. The results are depicted in Figure 1. Conclusion: HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A and positive control Actinomycin - D showed clear fragmented DNA after 24 hours treatments. There were no fragments observed in untreated cell control.

Example 8: GF/C Filter assay to check protein - DNA cross-links

The assay was designed as in reference, Proc. Natl. Acad. Sci. (U. S. A.), 1999, Vol. 96, 12168-12173, the disclosure of which is incorporated by reference for the teaching of the assay. GF/C glass fiber filter assays measure protein-DNA cross-links and are based on the selective binding of proteins to glass fiber filters in 0.4 M GuHCI. The assay was used to measure topoisomerase poisoning in vivo or in vitro. Cells were labeled with ³ H-dT (1 .0 μCi/mL) and after 36 hours, addition of extracts was carried out for 30 minutes. The medium was then removed and the cells were lysed with protein denaturants (500 μl of 6M GuHCI). The protein denaturants trap drug-stabilized topoisomerase-DNA strand passing intermediate (cleavable complexes) as irreversible protein - DNA cross-links. Then the lysate was transferred to a 1 .5 ml_ micro centrifuge tube containing a small stainless steel nut, the tube was capped securely, and the DNA was sheared by vortexing for 15 seconds. The lysate then was heated at 65 ⁰ C for 10 minutes to ensure denaturation and removal of noncovalently attached proteins from the DNA.

After cooling to room temperature, aliquots of the lysate were added to either 0.4 M or 4.0 M GuHCI solutions for filtration through GF/C glass fiber filters (Glass Fiber filter Papergrade C). In 4 M GuHCI (DNA binding condition), all nucleic acids bind the filter, and scintillation counting of the dried filter gives a measure of total labeled DNA in the aliquot. In 0.4 M GuHCI (protein binding condition), only protein binds to the glass fiber filter, and DNA was not retained unless it was cross-linked to protein. Comparison of the percentage of labeled DNA that was cross linked to protein on the filter in 0.4 M GuHCI to the radioactivity retained on the filter in 4 M GuHCI gives the

amount of DNA molecules cross-linked to protein. The topoisomerase subunits trapped in topoisomerase-DNA strand passing intermediates at the time of denaturation are covalently cross-linked to the substrate DNA and cause its retention on the glass fiber filter in 0.4 M GuHCI. Thus, the assay can be used to measure topoisomerase poisoning.

The results are depicted in Table 8.

Table 8: Effect on protein-DNA cross link

Conclusion:

The results indicate that the crude extract of Example 1 A and the enriched extract of Example 2A enhance the extent of protein-DNA cross link and therefore prevent replication and entry of cells into the apoptotic phase.

Example 9: CsCI gradient ultra centrifugation and dot blot of assay of topoisomerase Met

The assay was designed as in reference, J. Biol. Chem., 2001 , Vol. 276 (48), 44488- 44494, the disclosure of which is incorporated by reference for the teaching of the assay. The assay was used to check for topoisomerase I Ia in protein-DNA cross-linked complex. Cultured HEp-2 cells were treated with test samples and then lysed with 6 M GuHCI. The CsCI step gradients were prepared by the successive layering of 250 μl_ volumes of 1 .75 g/mL, 1 .63 g/mL, 1 .42 g/mL and 1 .32 g/mL into a small

ultracentrifuge tube. The lysates were loaded on a preformed CsCI gradient. Light mineral oil (Sigma) was used to fill the tube completely. The tubes were centrifuged in an SW41 rotor (Swinging Bucket Rotor) (Beckman Coulter) at 60,000 rpm for 2 hours at 20 ⁰ C. Fractions (125 μl_) were collected from the bottom of the tubes. Free proteins were left at the top of the gradient, and proteins that were covalently attached to DNA settled in the bottom. The fractions containing DNA were pooled and dialyzed to remove CsCI against TE buffer (10 mM Tris HCI, pH 7.4/ 1 mM EDTA). MgCI ₂ was added to the dialyzed sample to a final concentration of 5 mM, and the sample was treated with Deoxyribonuclease I (Roche Molecular

K) Biochemicals) 0.1 unit/ μl_ at 37 ⁰ C for 1 hour.

The Deoxyribonuclease I digested sample was then applied to a membrane (Amersham Pharmacia Biotech) with a dot blot device. The membrane was subsequently blocked with 3 % Skimmed Milk overnight at 4 ⁰ C. Membrane was washed with PBS three times for 5 minutes each and primary antibody of

15 topoisomerase I Ia was added in PBS containing 1 % BSA, 0.1 % Tween 20 was added to the blot along with the primary antibody and rocked gently at room temperature for 1 hour. The blot was washed 3 times with PBS for 5 minutes each. Secondary antibody (1 :5000) in 1 % BSA in PBS, 0.1 % Tween 20 was allowed to hybridize for 1 hour at room temperature. The bands were detected using 0 chromogenic substrate NBT-BCIP in alkaline phosphate buffer. The results are depicted in Table 9. Table 9 : Quantification of Topoisomerase ll-alpha in protein-DNA cross links

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30

Conclusion:

The data showed that the topoisomerase Il α was present in protein - DNA cross links formed in HEp-2 and M D A- M B -231 cells upon treatment with crude extract of Example 1 A and enriched extract of Example 2A.

Example 10: RNA extraction and amplification by reverse transcription - polymerase chain reaction (RT-PCR)

The assay was designed as in reference, Infection and Immunity, 1998, Vol.66, 4425- 30, the disclosure of which is incorporated by reference for the teaching of the assay. Cells were lysed in total RNA isolation reagent TriZol (Life Technologies), proteins were extracted with chloroform, and total RNA was precipitated with isopropanol. The RNA precipitate was washed with 70 % ethanol and resuspended in 50 μl_ of DEPC treated water. Reverse transcription was carried out to obtain cDNA with 200 units of avian reverse transcriptase using random hexamers. Primer sequences (synthesized by GIBCO BRL) were according to published information in Cell, 1998, Vol. 94, 491-501 . For PCR reactions 1 μL of the cDNA was added to a PCR reaction mixture consisting of 1 x PCR buffer, 2.5 pmol dNTP, 5 pmol of paired primers, 1 .25 units of Taq polymerase (Amersham Pharmacia Biotech) and distilled water in a total volume of 50 μL. The reaction mixture was placed in a PCR thermal cycler for cyclic reactions as follows: 95 ⁰ C for 5 minutes, 95 ⁰ C for 1 minute, annealing temperature for 1 minute, and 72 ⁰ C for 1 minute for a total of 35 cycles. PCR products were run on 1 .5 % Agarose gels, stained with ethidium bromide and photographed.

The results are depicted in Figure 2, Figure 3, Figure 4 and Figure 5.

Conclusions:

RT-PCR analysis of HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 8 hours showed upregulation of p53 when compared to untreated cell control, as indicated in Figure 2-A. GAPDH was used as internal cell control.

RT-PCR analysis of MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 8 hours did not show any significant level of

p53 expression, as indicated in Figure 2-B. GAPDH was used as internal cell control.

HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours showed downregulation of Bcl-2 when compared to untreated cell control, as indicated in Figure 3-A. GAPDH was used as internal cell control.

MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours showed downregulation of Bcl-2 when compared to untreated cell control, as indicated in Figure 3-B. GAPDH was used as internal cell control.

RT-PCR analysis of HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours showed upregulation of Bax gene level (Pro-apoptotic gene) when compared to untreated cell control, as indicated in Figure 4-A. GAPDH was used as internal cell control.

MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours did not show any significant induction of Bax gene expression, as indicated in Figure 4-B. GAPDH was used as internal cell control.

HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours did not show any significant expression of c-jun when compared to untreated cell control, as indicated in Figure 5-A. GAPDH was used as internal cell control.

MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours showed elevated level of c-jun expression when compared to untreated cell control, as indicated in Figure 5-B. GAPDH was used as internal cell control.

Example 11 : lmmunoblot analysis

Total cell lysates were prepared as in reference, MoI. Immunology, Vol.3(2), 1 15- 123, the disclosure of which is incorporated by reference for the teaching of the preparation of the cell lysates.

50 μg of proteins was separated on SDS 10 % polyacrylamide gel. The gel was transferred onto a nitrocellulose membrane (Hybond C+, Amersham life sciences.) at 20 V 120 mA for 1 hour and 30 minutes. Membrane was then washed 3 times with PBS and blocking was done with 3 % Skimmed Milk overnight at 4 °C. Membrane was washed with PBS three times for 5 minutes each and primary antibody was added (p53, BCI-2, cytochrome c with the specific concentration) in PBS containing 1 % BSA, 0.1 % Tween 20 was added to the blot along with the primary Antibody and rocked gently at room temperature for 1 hour. The blot was washed 3 times with PBS for 5 minutes each. Secondary Antibody (1 :5000) in 1 % BSA in PBS, 0.1 % Tween 20 was allowed to hybridize for 1 hour at room temperature. The bands were detected using chromogenic substrate NBT-BCIP in alkaline phosphate buffer.

The results are depicted in Figure 6, Figure 7 and Figure 8.

HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours showed elevated level of p53 protein expression when compared to untreated cell control, as indicated in Figure 6-A. Actin was used as internal cell control.

MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours did not show any expression of p53, as indicated in Figure 6-B. Actin was used as internal cell control.

HEp-2 cells treated with crude extract of Example 1 A and enriched extract of

Example 2A for 12 hours showed downregulation of Bcl-2 when compared to untreated cell control, as indicated in Figure 7-A. Actin was used as internal cell control.

MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 12 hours showed downregulation of Bcl-2 when compared to untreated cell control, as indicated in Figure 7-B. Actin was used as internal cell control.

HEp-2 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 16 hours showed higher level of cytochrome c release, as indicated in Figure 8-A. No cytochrome c release was observed in untreated cell control. Actin was used as internal cell control.

MDA-MB-231 cells treated with crude extract of Example 1 A and enriched extract of Example 2A for 16 hours showed higher level of cytochrome c release, as indicated in Figure 8-B. No cytochrome c release was observed in untreated cell control. Actin was used as internal cell control.

Example 12: Caspase 3 Activity assay (colorimetric assay)

Total cell lysates were prepared as in reference, MoI. Immunology, Vol.3(2), 1 15- 123, the disclosure of which is incorporated by reference for the teaching of the preparation of the cell lysates. The protein content of the supernatent was determined using Bio-Rad reagents. 50 μl_ of clear lysates were transferred to appropriate wells of the micro titer plate. 50 μl_ of extraction buffer was used as blank (Buffer Control). Recombinant Caspase - 3 was diluted as per the Kit protocols (Oncogene, Caspase - 3 Activity Assay). 50 μl_ of Assay buffer was added to each assay well followed by 10 μl_ of Substrate Conjugate. The plate was covered and incubated at 37 °C for 2 hours. The plate was then read at fluorescent plate reader with the specified excitation and emission wavelengths.The results are depicted in Table 10.

Table 10 :

Conclusion:

The caspase 3 activity assay revealed that the cells treated with crude extract of Example 1 A and enriched extract of Example 2A showed elevated levels of pro-

caspase-8 and effector-caspase-3 in HEp-2 and MDA-MB-231 cells as compared to untreated control cells.

Example 13: Cell Proliferation assay

The assay was designed as in reference, British Journal of Cancer, 2002, Vol.87, 98- 5 105, the disclosure of which is incorporated by reference for the teaching of the assay.

³ H-thymidine-incorporation was directly proportional to the number of dividing cells in culture.

After seeding the cells (at density, from 3000-5000 cells/well depending on cell type) K) in 180 μl_ in tissue culture grade 96 well plate, the cells were allowed to recover for

24 hours in humidified carbon dioxide (CO ₂ ) incubator (5 % CO ₂ ) at 37 ⁰ C.

20 μL of 10 mM stock of test sample in DMSO (dissolved first in DMSO and then in cell medium, final DMSO concentration should not exceed 0.5 %) in wells and plate was incubated for 48 hours in humidified CO ₂ incubator at 37 ⁰ C. After 48 hours, 15 medium from wells was removed and 0.5 μCi of ³ H-thymidine was added per well along with the cell medium to adjust total volume to 100 μL/well. The plate was incubated for 5 hours in humidified 5 % CO ₂ incubator at 37 ⁰ C. The cells were detached from the plate using 50 μL of 0.25 % trypsin. The cells on the 96 well glass-filter plate (Cat # 6005177, Unifilter-96, GF/B, Packard) were harvested using 0 Packard cell harvester and read on a Scintillation counter (Topcount, Packard, USA).

Percentage inhibition and IC ₅₀ were calculated in comparison with control values.

% CYTOTOXICITY = [CPM (TEST)/ CPM (CONTROL)]x100

The IC ₅ O was calculated by plotting a graph of % Cytotoxicity v/s Concentration and the results are as depicted in Table 11. 5

Cell lines used (Source: ATCC, USA)

1. Human non-small-cell lung carcinoma (H-460)

2. Human colon carcinoma (HCT-1 16)

3. Human breast carcinoma (ZR-75)

30 4. Human ovarian adenocarcinoma (A-2780)

On the basis of antiproliferation assay, Example 3-Fraction A and hence the compound of formula 1 was identified as the most potent of all samples submitted.

Example 14: Cell Proliferation Assay:

The assay was designed as in reference Proc. Natl. Acad. Sci. U. S. A., 2002, 99,

3609-3614, the disclosure of which is incorporated by reference for the teaching of the assay.

Test System The Panc-1 (pancreatic cancer), ACHN Renal cell carcinoma), H460 (Non-small cell lung cancer), Calu-1 (Lung cancer), HCT-1 16 (Colon cancer) and WI-38 (Normal primary lung cell line) cell line were procured from ATCC (American Tissue type

Culture Collection), USA.

Cell Counting Kit-8 (CCK-8) was procured from Dojindo Laoratories, Japan; Catalog number: CK-04-10000, tested and stored at 0 - 5 ⁰ C

Background: CCK-8 non-radioactive colorimetric assay was carried out to characterize the in vitro growth of human tumor cell lines as well as to test the cytotoxic activity of standard compounds.

Cell Counting Kit-8 (CCK-8) allows convenient assays using Dojindo's tetrazolium salt, WST-8 (8-(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-dis ulfophenyl)- 2H- tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon bioreduction in the presence of an electron carrier, 1 -methoxy PMS (1 -methoxy phenazinium methylsulfate). CCK-8 solution is added directly to the cells; no pre- mixing of components is required. CCK-8 is a sensitive non-radioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. The detection sensitivity of cell proliferation assays using WST-8 is higher than assays using the other tetrazolium salts such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] or MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4-sulfophenyl)-2H- tetrazolium salt]. Since the CCK-8 solution is very stable and it has little cytotoxicity, a longer incubation, such as 24 to 48 hours, is possible. Procedure

Different cancer cells (200 μl_) were seeded at a density of 3000 cells/well in a tissue culture grade 96 well plate and were allowed to recover for 24 hours in a humidified 5 % ± 0.2 CO ₂ incubator at 37 ⁰ C ± 0.5 ⁰ C. After 24 hours, was added 1 μl_ of 200X (200 times higher than required concentration is denoted as 200X) compound of formula 1 (dissolved in neat DMSO). The final DMSO concentration was 0.5 % in the wells. The plate was incubated for 48 hours in humidified 5 % ± 0.2 CO ₂ incubator at 37 ± 0.5 ⁰ C. After 48 hours, the plates were removed from the CO ₂ incubator and 5 μl_ of Cell counting Kit (CCK-8) per well was added. The plate was kept at 37 °C for 3 hours. The plates were thawed and allowed to come to room temperature. The absorbance at a wavelength of 450 nm was read. The percent cytotoxicity was calculated using the following formula:

Percent (Reading of Control -Reading of Treated cells) X 100

Cytotoxicity = Reading of Control

The results are as depicted in Table 12.

Table 12 :

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.