WO2012069972A1 - A pharmaceutical combination for the treatment of breast cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical combination comprising two cytotoxic antineoplastic agents, gemcitabine and carboplatin, and at least one cyclin dependent kinase (CDK) inhibitor; wherein said combination exhibits synergistic effects when used in the treatment of breast cancer, particularly triple negative breast cancer. The invention also relates to a method for the treatment of breast cancer, using a therapeutically effective amount of said combination.

Description

A PHARMACEUTICAL COMBINATION

FOR THE TREATMENT OF BREAST CANCER

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical combination for use in the treatment of triple negative breast cancer (TNBC) wherein said combination exhibits a synergistic effect. The pharmaceutical combination comprises two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and at least one cyclin dependent kinase (CDK) inhibitor represented by a compound of formula I (as described herein) or a pharmaceutically acceptable salt thereof. The present invention also relates to a method of treating breast cancer in a subject comprising administering to the subject a therapeutically effective amount of two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts and at least one cyclin dependent kinase (CDK) inhibitor selected from the compounds of formula I (as described herein) or a pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION

Cancer is a general term used to describe diseases in which abnormal cells divide without control. Cancer cells can invade adjacent tissues and can spread through the bloodstream and lymphatic system to other parts of the body. There are different types of cancers such as the bladder cancer, breast cancer, colon cancer, rectal cancer, head and neck cancer, endometrial cancer, kidney (renal cell) cancer, leukemia, small cell lung cancer, non- small cell lung cancer, pancreatic cancer, prostate cancer, thyroid cancer, skin cancer, Non- Hodgkin's Lymphoma and melanoma. Currently there are many treatments available for cancer than ever before, including chemotherapy, radiation, surgery, hormonal therapy, immune therapy and gene therapy. Chemotherapy is the most routinely used treatment for cancer.

The most widely used chemotherapeutic agents (the antineoplastic agents) include paclitaxel, docetaxel, doxorubicin, etoposide, carboplatin, cisplatin, topotecan and gemcitabine. These antineoplastic agents have been successfully used for the treatment of different cancers. However, in due course of time, some cancer patients have been found to develop resistance to monotherapy involving use of such standard antineoplastic agents. Tolerance or resistance to a drug represents a major impediment to successful treatment. Such resistance is often considered as either intrinsic (i.e. present at the onset of treatment) or acquired (i.e. occurs during the course of chemotherapy). A study involving exposure of human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of doxorubicin reported appearance of a new cell line (NCI-H460/R) that was resistant to doxorubicin and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin (J. Chemother., 2006, 18, 1, 66-73). Gemcitabine was considered to be the most clinically active drug for the treatment of pancreatic cancer, however it failed to significantly improve the condition of pancreatic cancer patients because of the pre-existing or acquired chemo resistance of the tumor cells to the drug (Oncogene, 2003, 22, 21, 3243-51).

Another problem observed or prevalent in the cancer treatment is the severe toxicity associated with most of the antineoplastic agents. Despite the incidence of resistance and severe toxicity associated with the conventional antineoplastic agents e.g. gemcitabine and paclitaxel, these agents will continue to be important in the cancer treatment because they have the ability to reduce tumor mass. In order to improve the response rate and prevent toxicity associated with the conventional antineoplastic agents, new therapeutic approaches are being evaluated.

One such approach is directed to a protocol involving combining different anticancer agents. An optimal combination chemotherapy protocol may result in increased therapeutic efficacy, decreased host toxicity, and minimal or delayed drug resistance. When drugs with different toxicities are combined, each drug can be used at its optimal dose, helping minimise intolerable side effects. Some of the antineoplastic agents have been found to be synergistically effective when used in combination with other anticancer agents than when used as a monotherapy.

Cyclophosphamide and 5-fluorouracil act synergistically in ovarian clear cell adenocarcinoma cells (Cancer Lett., 2001, 162, 1, 39-48). Combination chemotherapy can also be advantageously used for treating cancers in advanced stages which are difficult to treat with monotherapy, radiation or surgical treatment, for example, a combination of paclitaxel and gemcitabine has been reported for the treatment of metastatic nonsmall cell lung cancer (Cancer, 2006, 107, 5, 1050-1054). Gemcitabine and carboplatin combination chemotherapy was relatively safe and effective for treating elderly patients with non-small cell lung cancer. (Cancer Res. Treat., 2008, 40, 116-120). Gemcitabine plus carboplatin combination is active in advanced TCC (transitional cell carcinoma) with acceptable toxicity (BMC Cancer, 2007, 7, 98). Treatment with gemcitabine and carboplatin significantly improves the progression-free survival of patients with platinum-sensitive recurrent ovarian cancer (Int. J. Gynecol. Cancer, 2005, 15 (Suppl. 1), 36^-1). Recently, combination of one or more standard antineoplastic agents such as paclitaxel, cisplatin etc. with a molecularly targeted anticancer agent for the treatment of cancer has been tried out to improve drug response rates and to address resistance to the antineoplastic agents. Molecularly targeted agents e.g. imatinib mesylate, flavopiridol etc. modulate proteins such as kinases whose activities are more specifically associated with cancerous cells. Researches over a long period of time have proven that the members of the cyclin-dependent kinase (CDK) family play key roles in various cellular processes. There are 11 members of the CDK family known till now. Among these, CDK1, CDK2, CDK3, CDK4 and CDK6 are known to play important roles in the cell cycle (Adv. Cancer Res., 1995, 66, 181-212). CDKs are activated by forming noncovalent complexes with cyclins such as A- type, B- type, C- type, D-type (Dl, D2, and D3), and E-type cyclins. Each isozyme of this family is responsible for particular aspects (cell signaling, transcription, etc.) of the cell cycle, and some of the CDK isozymes are specific to certain kinds of tissues. Aberrant expression and overexpression of these kinases are evidenced in many disease conditions. A number of compounds having potentially useful CDK inhibitory properties have been developed and reported in the literature.

Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to reach clinical trial. Flavopiridol has been found to potentiate synergistically the cytotoxic response of the conventional cytotoxic antineoplastic agents in a variety of cancer cell-lines. For example, combined docetaxel and flavopiridol treatment for lung cancer cells has been reported in Radiother. Oncol., 2004, 71, 2, 213-221 and for the treatment of gastric cancer in Mol. Cancer Ther., 2003, 2, 6, 549-555. PCT publication WO2008139271 discloses the combinations of a CDK inhibitor, (+)-ira«s-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2 -hydroxy methyl -l-methyl-pyrrolidin-3-yl)-chromen -4-one hydrochloride with cytotoxic neoplastic agents such as doxorubicin, docetaxel, paclitaxel and gemcitabine for the treatment of non- small cell lung carcinoma and pancreatic cancer.

Although various treatment options are available for the treatment of cancers, this disease still remains one of the most fatal diseases. Although, all the types of cancers are fatal, breast cancer still remains a type of fatal cancer. In fact, in women, breast cancer is among the most common cancers and is the fifth most common cause of cancer deaths. Different forms of breast cancers can have remarkably different biological characteristics and clinical behavior. Thus, classification of a patient's breast cancer has become a critical component for determining a treatment regimen. Breast cancer patients fall into three main groups: (i) those with hormone receptor-positive tumors who are managed with a number of estrogen receptor (ER)- targeted therapy options ± chemotherapy;

(ii) those with HER2+ tumors, who will, in addition, receive HER2-directed therapy with trastuzumab or, in some situations, lapatinib; and

(iii) those with hormone receptor [ER and progesterone receptor (PR)] -negative and

HER2) breast cancers, for whom chemotherapy is the only modality of systemic therapy available.

Currently, trastuzumab has been developed as a targeted therapy for breast cancer patients. Studies have shown that the expression profiles of breast cancer display a systematic variation and allow classification of breast cancer into five main groups, two of them ER+ (luminal A and B) and three ER- groups [normal breast-like, ERBB2 (also known as HER2) and 'basal-like']. It has been shown that the basal-like group is enriched for tumors that lack expression of hormone receptors and of HER2 and has a more aggressive clinical behavior, a distinctive metastatic pattern and a poor prognosis despite responding to conventional neoadjuvant and adjuvant chemotherapy regimens. Based on the above it is clear that the interest in triple-negative cancers stems from (i) the lack of tailored therapies for this group of breast cancer patients and (ii) overlap with the profiles of basal-like cancers (Histopathology, 2008, 52, 108-118).

Triple-negative breast cancer (TNBC) i.e., tumors that are estrogen receptor (ER)- negative and progesterone receptor (PR)-negative and do not overexpress human epidermal growth factor receptor 2 (HER2) account for approximately 15 % of breast cancers, with approximately 170,000 cases reported worldwide in 2008. Triple-negative cancers are significantly more aggressive (metastatic) than tumors pertaining to other molecular subgroups. TNBC does not express estrogen (ER), progesterone (PR) and HER2 receptors, therefore, they are resistant to currently available targeted treatment, including hormonal and HER2-targeted therapies. Patients with basal-like or triple negative breast cancers have a significantly shorter survival following the first metastatic event when compared with those with non-basal-like/non-triple negative patients. A vast majority of tumors arising in BRCA1 germ-line mutation carriers have morphological features similar to those described in basal- like cancers and they display a triple negative and basal like phenotype.

Triple-negative breast cancer constitutes one of the most challenging groups of breast cancers. The only systemic therapy currently available for patients with such cancers is chemotherapy. However, the survival of patients with such tumors is still poor and their management may, therefore, require a more aggressive intervention. As a result the development of targeted therapies for triple-negative cancers is of considerable importance. Recent trials have shown that poly (ADP-ribosyl)ation polymerase (PARP) inhibitor, BSI- 201 (currently known as Iniparib a compound developed by Sanofi-Aventis) is highly effective in TNBC (Maturitas, 2009, 63, 269-274). Also TNBC is characterized by elevated levels of PARP. These characteristics have suggested that PARP inhibition might be able to potentiate the effects of chemotherapy-induced DNA damage in TNBC (Community Oncology, 2010, 7, 5, 2, 7-10; Clinical Advances in Hematology and Oncology, 7, 7, 441- 443).

Although triple-negative breast cancers are reported to respond to chemotherapy, survival of patients with such tumors is still poor and their management may therefore require a more aggressive alternative intervention. Thus, the development of biologically informed systemic therapies and targeted therapies for triple-negative breast cancers are of paramount importance and may prove to be achievable by understanding the complexity of this heterogeneous group of tumors and using combination therapy (Histopathology, 2008, 52, 108-118).

In view of the above discussion and considering that treatment options for treating triple negative breast cancer are very limited, a need remains for additional treatment options and methods for treating TNBC. SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a pharmaceutical combination for use in the treatment of triple negative breast cancer; said combination comprising two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and a cyclin dependent kinase (CDK) inhibitor selected from the compound of formula I (as described herein) or a pharmaceutically acceptable salt thereof.

In another aspect, the pharmaceutical combination of the present invention exhibits synergistic effect in the treatment of triple negative breast cancer.

In another aspect, the present invention relates to a pharmaceutical combination for use in the treatment of triple negative breast cancer comprising two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and a cyclin dependent kinase (CDK) inhibitor selected from the compound of formula I (as described herein) or a pharmaceutically acceptable salt thereof; wherein the two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and the CDK inhibitor are administered sequentially. In another further aspect, the present invention relates to a method of treating triple negative breast cancer in a subject comprising administering to the subject a therapeutically effective amount of two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and a therapeutically effective amount of a cyclin dependent kinase (CDK) inhibitor selected from the compounds of formula I (as described herein) or a pharmaceutically acceptable salt thereof.

In a still further aspect, the present invention relates to use of the pharmaceutical combination for the manufacture of a medicament for treating triple negative breast cancer.

Other aspects and further scope of applicability of the present invention will become apparent from the detailed description to follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Effect of Compound A on colony formation in breast cancer cell lines (MDA- MB-231, MDA-MB-468 and MCF-7)

Figure 2: Effect of Compound A on MCTS formation in MCF-7 breast cancer cell line

Figure 3A: Time dependent effect of Compound A on cell cycle progression and apoptosis in MCF-7 (Her2-, BRCA +/- allelic loss) cell line

Figure 3B: Time dependent effect of Compound A on cell cycle progression and apoptosis in MDA-MB-231 cell line

Figure 4: Expression of antiapoptotic protein Bcl-2 in MCF-7 and MDA-MB-231 cell lines treated with Compound A

Figure 5A: Effect of Compound A on MDA-MB-231 cell line (different phases of the cell cycle)

Figure 5B: Effect of Compound A on MDA-MB-468 cell line

Figure 5C: Effect of BSI-201 on MDA-MB-231 and MDA-MB-468 cell lines

Figure 6A: Cyclin Dl level in various breast cancer cell lines

Figure 6B: Effect of Compound A on MCF-7 cell cycle proteins and CDK4 kinase activity

Figure 7: Effect of Compound A on PARP enzyme activity in breast cancer cell lines

(MDA-MB-231 and MDA-MB-468) as measured by PAR polymers

Figure 8: Effect of Compound A (24 h) on PARP and cell cycle proteins in MDA-MB- 231 and MDA-MB-468 triple negative breast cancer cell lines Figure 9: Effect of Compound A on HIF-Ι inhibition in the U251 HRE and U251 pGL3 cell lines Figure 10: Effect of Compound A on VEGF inhibition using the VEGF reporter gene based assay

Figure 11A: Effect of Compound A on the migration of BT-549 breast cancer cell line Figure 11B: Effect of Compound A on the migration of MDA-MB-231 breast cancer cell line

Figure 11 C: Effect of Compound A on the migration of MCF-7 breast cancer cell line

Figure 12: Effect of Compound A on endothelial tube formation as observed in

Endothelial Cell Tube Formation Assay

Figure 13A: Effect of the combination of Gemcitabine and Carboplatin together for 24 h followed by Compound A (IC₅₀) for 72 h in MD A-MB -231 cell line

Figure 13B: Effect of the combination of various concentrations of Gemcitabine and

Carboplatin together for 24 h followed by Compound A (IC5₀) for 96 h in

MDA-MB-231 cell line

Figure 14A: Effect of the combination of Gemcitabine (ICio) for 6 h followed by

Carboplatin (ICio) for 24 h followed by Compound A (IC₃₀ IC5₀) for 72 h in

MDA-MB-231 cell line

Figure 14B: Effect of the combination of Gemcitabine (ICio) for 6 h followed by

Carboplatin (ICio) for 24 h followed by Compound A (IC₃₀ IC5₀) for 96 h in

MDA-MB-231 cell line

Figure 15A: Effect of the combination of Gemcitabine (ICio) for 6 h followed by

Carboplatin (ICio) for 24 h followed by BSI-201 (IC30/IC50) for 72 h in MDA- MB-231 cell line

Figure 15B: Effect of the combination of Gemcitabine (IC₁₀) for 6 h followed by

Carboplatin (1C_W) for 24 h followed by BSI-201 (lC₃₀flC₅₀) for 96 h in MDA- MB-231 cell line

Figure 16: Average tumor growth profile of human breast cancer (MDA-MB-231) xenograft in triple drug combination study

Figure 17: Average percent growth inhibition in the human breast cancer (MDA-MB- 231) xenograft model as seen in the triple drug combination study Figure 18: Average percent weight profile of human breast cancer (MDA-MB-231) xenograft model in SCID mice in triple drug combination study DETAILED DESCRIPTION OF THE INVENTION

It has now been found that the pharmaceutical combination of the present invention, which comprises two cytotoxic antineoplastic agents, gemcitabine and carboplatin, or their pharmaceutically acceptable salts and a CDK inhibitor selected from the compounds of formula I (as described herein) or a pharmaceutically acceptable salt thereof; exhibits synergistic effect when used in the treatment of triple negative breast cancer.

In particular, the present invention provides a method of treating, or managing triple negative breast cancer in a subject comprising administering to said subject a therapeutically effective amount of two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and a therapeutically effective amount of a cyclin dependent kinase (CDK) inhibitor selected from the compounds of formula I (as described herein) or a pharmaceutically acceptable salt thereof.

The CDK inhibitor comprised in the pharmaceutical combination of the present invention is selected from a compound of formula I as described herein below. The CDK inhibitors represented by the following formula I are disclosed in PCT Publication No. WO2004004632 (corresponding to U.S. Patent 7,272,193) and PCT Publication No. WO2007148158, which are incorporated herein by reference. The compounds of formula I are CDK inhibitors, which inhibit proliferation of different cancer cells. The compounds of formula I as used in the present invention are effective against various solid and hematological malignancies. The inventors of the present invention observed that combining CDK inhibitors, the compounds of formula I with cytotoxic antineoplastic agents namely gemcitabine and carboplatin resulted in an increase in apoptosis, or programmed cell death.

The CDK inhibitors used in the present invention are selected from the compounds represented by the following formula I,

wherein Ar is a phenyl group, which is unsubstituted or substituted by 1 , 2, or 3 identical or different substituents selected from : halogen selected from chloro, bromo, fluoro or iodo; nitro, cyano, Ci-C4-alkyl, trifluoromethyl, hydroxy, Ci-C4-alkoxy, carboxy, d-C₄- alkoxycarbonyl, CONH₂ or NR1R2 ;

wherein Ri and R2 are each independently selected from hydrogen or Ci-C4-alkyl.

The compounds of formula I, which includes enantiomerically pure forms thereof, may be prepared according to the methods disclosed in PCT Publication No. WO2004004632 and PCT Publication No. WO2007148158, which are incorporated herein by reference.

The general process for the preparation of compounds of Formula (I), or a pharmaceutically acceptable salt thereof, comprises the following steps:

(a) treating the resolved enantiomerically pure (-)-trans enantiomer of the intermediate compound of Formula VIA,

VIA

with acetic anhydride in the presence of a Lewis acid catalyst to obtain a resolved acetylated compound of Formula VIIA,

VI IA

(b) reacting the resolved acetylated compound of Formula VIIA with an acid of Formula ArCOOH or an acid chloride of Formula ArCOCl or an acid anhydride of Formula (ArCO)₂0 or an ester of Formula ArCOOCH₃, wherein Ar is as defined hereinabove in reference to the compound of Formula (I), in the presence of a base and a solvent to obtain a resolved compound of Formula VIII A;

VI I IA

(c) treating the resolved compound of Formula VIIIA with a base in a suitable solvent to obtain the corresponding resolve -diketone compound of Formula IXA;

IXA

wherein Ar is as defined above;

(d) treating the resolved β-diketone compound of Formula IXA with an acid such as hydrochloric acid to obtain the corresponding cyclized compound of Formula XA,

XA

(e) subjecting the compound of Formula XA to dealkylation by heating it with a dealkylating agent at a temperature ranging from 120-180 °C to obtain the (+)-trans enantiomer of the compound of Formula (I) and, optionally, converting the subject compound into its pharmaceutically acceptable salt.

The Lewis acid catalyst utilized in the step (a) above may be selected from: BF_3, Et₂0, zinc chloride, aluminium chloride and titanium chloride.

The base utilized in the process step (b) may be selected from triethylamine, pyridine and a DCC-DMAP combination (combination of N, N' -dicyclohexyl carbodiimide and 4- dimethylaminopyridine) . It will be apparent to those skilled in the art that the rearrangement of the compound of Formula VIIIA to the corresponding β-diketone compound of Formula IXA is known as a Baker-Venkataraman rearrangement (J. Chem. Soc, 1933, 1381 and Curr. Set, 1933, 4, 214).

The base used in the process step (c) may be selected from: lithium hexamethyl disilazide, sodium hexamethyldisilazide, potassium hexamethyldisilazide, sodium hydride and potassium hydride. A preferred base is lithium hexamethyl disilazide.

The dealkylating agent used in process step (e) for the dealkylation of the compound of Formula IXA may be selected from: pyridine hydrochloride, boron tribromide, boron trifluoride etherate and aluminium trichloride. A preferred dealkylating agent is pyridine hydrochloride.

Preparation of the starting compound of Formula VIA involves reacting l-methyl-4- piperidone with a solution of 1,3,5-trimethoxybenzene in glacial acetic acid, to yield 1- methyl-4-(2,4,6-trimethoxyphenyl)-l,2,3,6-tetrahydropyridine, which is reacted with boron trifluoride diethyl etherate, sodium borohydride and tetrahydrofuran to yield l-methyl-4- (2,4,6-trimethoxyphenyl)piperidin-3-ol. Conversion of l-methyl-4-(2,4,6- trimethoxyphenyl)piperidin-3-ol to the compound of Formula VIA involves converting the hydroxyl group present on the piperidine ring of the compound, l-methyl-4-(2,4,6- trimethoxyphenyl)piperidin-3-ol to a leaving group such as tosyl, mesyl, triflate or halide by treatment with an appropriate reagent such as p-toluenesulfonylchloride, methanesulfonylchloride, triflic anhydride or phosphorous pentachloride in the presence of oxygen nucleophiles such as triethylamine, pyridine, potassium carbonate or sodium carbonate_, followed by ring contraction in the presence of oxygen nucleophiles such as sodium acetate or potassium acetate in an alcoholic solvent such as isopropanol, ethanol or propanol.

In an embodiment the CDK inhibitor is a compound of formula I wherein the phenyl group is substituted by 1, 2, or 3 identical or different substituents selected from: halogen selected from chlorine, bromine, fluorine or iodine; Ci-C4-alkyl and trifluoromethyl.

In another embodiment, the CDK inhibitor is a compound of formula I wherein the phenyl group is substituted by 1, 2, or 3 halogens selected from chlorine, bromine, fluorine or iodine.

In another embodiment, the CDK inhibitor is a compound of formula I wherein the phenyl group is substituted by chlorine. In a further embodiment, the CDK inhibitor represented by compound of formula I is (+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)- chromen-4-one or its pharmaceutically acceptable salt.

In still further embodiment, the CDK inhibitor represented by compound of formula I is (+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3- yl)-chromen-4-one hydrochloride (designated herein as compound A).

In another embodiment, the CDK inhibitor is a compound of formula I wherein the phenyl group is disubstituted with a chloro and a trifluoromethyl group.

In a further embodiment, the CDK inhibitor represented by compound of formula I is (+)-ira«i-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2 -hydro xymethyl-1 -methyl -pyrrolidin-3-yl)-chromen-4-one; or its pharmaceutically acceptable salt.

In still further embodiment, the CDK inhibitor represented by compound of formula I is (+)-ira«i-2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l- methyl -pyrrolidin-3-yl)-chromen-4-one hydrochloride (designated herein as compound B).

In an embodiment, the CDK inhibitor represented by a compound of formula I is an antiangiogenic agent.

In an embodiment, the CDK inhibitor represented by a compound of formula I is a HIF-Ι inhibitor. In an embodiment, the CDK inhibitor represented by a compound of formula I is a VEG-F inhibitor. In an embodiment, the CDK inhibitor represented by a compound of formula I is a PARP enzyme inhibitor.

The manufacture of the compounds of formula I, which may be in the form of pharmaceutically acceptable salts, and the manufacture of oral and/or parenteral pharmaceutical composition containing the above compounds are disclosed in PCT Publication No. WO2004004632 (corresponding to U.S. Patent 7,272,193) and PCT Publication No. WO2007148158. These PCT Publications disclose that the CDK inhibitors represented by formula I inhibit proliferation of different cancer cells. As indicated herein above the CDK inhibitors of formula I may be used in the form of their salts. Preferred salts of the compounds of formula I include hydrochloride salt, methanesulfonic acid salt and trifluoroacetic acid salt.

The compounds of formula I contain at least two chiral centers and hence exist in the form of two different optical isomers (i.e. (+) or (-) enantiomers). All such enantiomers and mixtures thereof including racemic mixtures are included within the scope of the invention. The enantiomers of the compound of formula I can be obtained by methods disclosed in PCT Publication No. WO2004004632, WO2008007169 and WO2007148158 or the enantiomers of the compound of formula I can also be obtained by methods well known in the art, such as chiral HPLC and enzymatic resolution. The term "enantiomerically pure" describes a compound which is present in an enantiomeric excess (ee) of greater than 95 %. In another embodiment, the enantiomeric excess is greater than 97%. In still another embodiment, the enantiomeric excess is greater than 99%. The term "enantiomeric excess" describes the difference between the amount of one enantiomer and the amount of another enantiomer that is present in the product mixture.

Alternatively, the enantiomers of the compounds of formula I can be synthesized by using optically active starting materials. Thus, the definition of the compounds of formula I is inclusive of all possible stereoisomers and their mixtures. The definition of the compound of formula I includes the racemic forms and the isolated optical isomers having the specified activity.

The two cytotoxic antineoplastic agents used in the pharmaceutical combination of the present invention are selected from gemcitabine and carboplatin, which are commercially available.

Gemcitabine is the generic name assigned to 2'-deoxy-2',2'-difluorocytidine. It is commercially available as the monohydrochloride salt, and as the β-isomer. Gemcitabine is disclosed in U.S. Pat. Nos. 4,808,614 and 5,464,826, which are incorporated herein by reference for their teaching of how to synthesize and use gemcitabine for treating susceptible cancers. The commercial formulation of gemcitabine hydrochloride as a single agent is indicated as first-line treatment for patients with locally advanced or metastatic adenocarcinoma of the pancreas or lung cell carcinoma (NSCLC), and is commonly used in patients previously treated with 5-fluorouracil.

Carboplatin is the generic name assigned to cis-diamine(l,l- cyclobutanedicarboxylato)platinum. Carboplatin was discovered and developed at the Institute of Cancer Research in London. Carboplatin is disclosed in U.S. Pat. No. 4,657,927, which is incorporated herein by reference for its teaching of how to synthesize and use carboplatin for treating susceptible cancers. Carboplatin kills cancer cells by binding to DNA and interfering with the cell's repair mechanism, which eventually leads to cell death. It is classified as an alkylating agent. It is considered a "second-generation" platinum agent. Carboplatin differs chemically from cisplatin by being a bigger molecule, with a dicarboxylate ligand. This slows the metabolic breakdown of the agent (it stays in the body longer) and reduces the rate of formation of toxic by-products. Carboplatin is used to treat ovarian cancer. Carboplatin is also used for other types of cancer, including lung, head and neck, endometrial, esophageal, bladder, breast, and cervical; central nervous system or germ cell tumors; osteogenic sarcoma; and as preparation for a stem cell or bone marrow transplant.

The general terms used hereinbefore and hereinafter preferably have a meaning within the context of this disclosure the following meanings, unless otherwise indicated:

The term "antineoplastic agent" is synonymous to "chemo therapeutic agent" or "anticancer agent" and refers to a therapeutic agent, which acts by inhibiting or preventing the growth of neoplasms. The term "antineoplastic agent" or "anticancer agent" in general refers to compounds that prevent cancer cells from multiplying (i.e. anti-proliferative agents). In general, the antineoplastic agent(s) fall into two classes, anti-proliferative cytotoxic and anti-proliferative cytostatic. Antiproliferative cytotoxic agents prevent cancer cells from multiplying by: (1) interfering with the cell's ability to replicate DNA and (2) inducing cell death and/or apoptosis in the cancer cells. Anti-proliferative cytostatic agents act via modulating, interfering or inhibiting the processes of cellular signal transduction which regulate cell proliferation. In the present invention the antineoplastic agents comprised in the pharmaceutical combination of the present invention are cytotoxic agents and hence are referred to as cytotoxic antineoplastic agents.

As used herein, the term "combination" or "pharmaceutical combination", means the combined administration of the two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharamceutically acceptable salts; and the CDK inhibitor (the compound of formula I); which therapeutic agents may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a synergistic effect.

As used herein, the term "synergistic" means that the effect achieved with the methods and combinations of this invention is greater than the sum of the effects that result from using the antineoplastic agents or their pharmaceutically acceptable salts, and a CDK inhibitor, the compound of formula I or a pharmaceutically acceptable salt thereof, separately. Advantageously, such synergy provides greater efficacy at the same doses, and/or prevents or delays the build-up of multi-drug resistance.

A "therapeutically effective amount", in reference to the treatment of triple negative breast cancer, refers to an amount capable of invoking one or more of the following effects in a subject receiving the combination of the present invention: (i) inhibition, to some extent, of tumor growth, including, slowing down and complete growth arrest; (ii) reduction in the number of cancerous cells; (iii) reduction in tumor size; (iv) inhibition (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (v) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (vi) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; and/or (vii) relief, to some extent, of one or more symptoms associated with triple negative breast cancer.

As used herein, the terms "manage", "managing" and "management" refer to the beneficial effects that a subject or a patient derives from the pharmaceutical combination of the present invention when administered to said patient or subject so as to prevent the progression or worsening of TNBC.

As used herein the term "triple negative breast cancer(s)" or "TNBC" encompasses carcinomas of differing histopathological phenotypes. For example, certain TNBC are classified as "basal-like" ("BL"), in which the neoplastic cells express genes usually found in normal basal/myoepithelial cells of the breast, such as high molecular weight basal cytokeratins (CK, CK5/6, CK14, CK17), vimentin, p-cadherin, ccB crystallin, fascin and caveolins 1 and 2. Certain other TNBC, however, have a different histopathological phenotype, examples of which include high grade invasive ductal carcinoma of no special type, metaplastic carcinomas, medullary carcinomas and salivary gland-like tumors of the breast. The TNBC for the treatment of which the pharmaceutical combination of the present invention is provided may be non-responsive or refractory TNBC.

The phrase "non-responsive/refractory" as used herein, is used to describe subjects or patients having triple negative breast cancer(TNBC) having been treated with currently available cancer therapies for the treatment of TNBC such as chemotherapy, radiation therapy, surgery, hormonal therapy and/or biological therapy/immunotherapy wherein the therapy is not clinically adequate to treat the patients such that these patients need additional effective therapy, e.g., remain unsusceptible to therapy. The phrase can also describe subjects or patients who respond to therapy yet suffer from side effects, relapse, develop resistance, etc. In various embodiments, "non-responsive/refractory" means that at least some significant portions of the cancer cells are not killed or their cell division arrested. The determination of whether the cancer cells are "non-responsive/refractory" can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context. A cancer is "non- responsive/refractory" where the number of cancer cells has not been significantly reduced, or has increased. As used herein the term "treatment cycle" refers to a time period during which a recurring sequence of administration of cytotoxic antineoplastic agents or their pharmaceutically acceptable salts, and a CDK inhibitor i.e. the compound of formula I or a pharmaceutically acceptable salt thereof, is carried out.

The term "apoptosis" refers to a type of cell death in which a series of molecular steps in a cell leads to its death. This is the body's normal way of getting rid of unneeded or abnormal cells. The process of apoptosis may be blocked in cancer cells. Also called programmed cell death. (Dictionary of cancer terms, National Cancer Institute)

As used herein the term "increasing apoptosis" is defined as an increase in the rate of programmed cell death, i.e. more cells are induced into the death process as compared to exposure (contact) with either the combination of only the antineoplastic agents or the CDK inhibitor alone.

The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

In one embodiment, the present invention relates to a pharmaceutical combination for use in the treatment of triple negative breast cancer wherein said combination comprises two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts and at least one cyclin dependent kinase (CDK) inhibitor selected from the compounds of formula I (as described herein) or a pharmaceutically acceptable salt thereof.

In an embodiment, the pharmaceutical combination comprising the CDK inhibitor i.e. the compound of formula I and the cytotoxic antineoplastic agents as described herein, encompass those which permit a separate administration, which can be sequential or spaced out over a period of time so as to obtain maximum efficacy of the combination. Thus, the pharmaceutical combination may be administered separately or spaced out over a period of time for an effective cancer treatment.

In an embodiment of the present invention, the cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts are administered prior to administration of the CDK inhibitor i.e. the compound of formula I or a pharmaceutically acceptable salt thereof.

In an embodiment, the CDK inhibitor comprised in the pharmaceutical combination of the present invention is selected from the compound A or compound B.

In an embodiment, the therapeutic agents i.e. the cytotoxic antineoplastic agents and the CDK inhibitor comprised in the combination may have to be administered by different routes, because of their different physical and chemical characteristics. For example, the CDK inhibitors of the compound of formula I may be administered either orally or parenterally to generate and maintain good blood levels thereof, while the antineoplastic agents may be administered parenterally, by intravenous, subcutaneous or intramuscular route.

For oral use, the CDK inhibitors of the compound of formula I may be administered, for example, in the form of tablets or capsules, powders, dispersible granules, or cachets, or as aqueous solutions or suspensions. In the case of tablets for oral use, carriers which are commonly used include lactose, corn starch, magnesium carbonate, talc, and sugar, and lubricating agents such as magnesium stearate are commonly added. For oral administration in capsule form, useful carriers include lactose, corn starch, magnesium carbonate, talc and sugar.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient (the antineoplastic agents or the CDK inhibitor) are usually employed, and the pH of the solutions should be suitably adjusted and buffered.

In an embodiment, the sterile solutions of the active ingredient used are prepared in saline or distilled water.

In an embodiment, the present invention relates to a method of treating of triple negative breast cancer in a subject comprising administering to the subject a therapeutically effective amount of the two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts in combination with a therapeutically effective amount of a CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

Accordingly, in the method of the present invention, triple negative breast cancer is treated in a subject by administering to the subject a therapeutically effective amount of the two cytotoxic antineoplastic agents effective to treat the cancer, in combination with a therapeutically effective amount of a CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof, wherein the combined use of the therapeutic agents exhibits a synergistic effect .

In an embodiment, a method of treating triple negative breast cancer in a subject comprising administering to the subject a therapeutically effective amount of the combination of the cytotoxic antineoplastic agents, gemcitabine and carboplatin and a therapeutic amount of the CDK inhibitor represented by a compound of formula I, wherein the cytotoxic antineoplastic agents, gemcitabine and carboplatin and the CDK inhibitor are administered sequentially. In another embodiment, the method of treating triple negative breast cancer in a subject comprising administering to the subject a therapeutically effective amount of the cytotoxic antineoplastic agents, gemcitabine and carboplatin prior to administration of the CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

In an embodiment, the method of treating triple negative breast cancer in a subject comprises administering to the subject gemcitabine and carboplatin sequentially followed by the administration of the CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

In an embodiment, the method of treating triple negative breast cancer in a subject comprises administering to the subject gemcitabine and carboplatin are administered simultaneously followed by the administration of the CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

In an embodiment, the CDK inhibitor used in the method of treating triple negative breast cancer is selected from the compound A or compound B.

In an embodiment, the present invention relates to use of a pharmaceutical combination for the manufacture of a medicament for use in the treatment of triple negative breast cancer, wherein said pharmaceutical combination comprises two cytotoxic antineoplastic agents, gemcitabine and carboplatin and a CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof.

The actual dosage of the anticancer agents contained in the combination may be varied depending upon the requirements of the patient and the severity of the condition being treated. Generally, treatment is initiated with smaller doses, which are less than the optimum dose of the compound. Thereafter, the dose of each anticancer agent is increased by small amounts until the optimum effect under the circumstances is reached. However, the amount of each anticancer agent in the pharmaceutical combination will typically be less than an amount that would produce a therapeutic effect if administered alone. For convenience, the total daily dose may be divided and administered in portions during the day if desired. In an embodiment, the two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts, and a CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof are administered sequentially in injectable forms, such that the cytotoxic antineoplastic agents are administered in a synergistically effective dose ranging from 10 mg to 2000 mg each, and the CDK inhibitor is 2 administered in the CDK inhibitor is administered in a dose ranging from 5 mg/m /day to 1000 mg/m²/day, particularly in a dose ranging from 9 mg/m²/day to about 259 mg/m²/day.

In an embodiment, the pharmaceutical combination provided for use in the treatment of triple negative breast cancer is administered to a subject in need thereof, for six to eight treatment cycles, particularly six treatment cycles; two consecutive treatment cycles comprising the following steps:

i) a single dose administration of the pharmaceutical combination of gemcitabine, carboplatin and Compound A on day one of the treatment cycle;

ii) from second day, administration of one dose per day of Compound A for four consecutive days;

iii) a two- day interval wherein no drug (anticancer agent) is administered;

iv) optional administration of Compound A for five consecutive days followed by two- day interval with no drug (anticancer agent) administration;

v) optionally repeating step iv); and

vi) repeating steps i) to v) as a second treatment cycle, after an interval of three weeks from the beginning of step i).

In an embodiment, the pharmaceutical combination is administered to a subject in need thereof, for two to six treatment cycles, before surgery or after surgery or partially before and partially after surgery.

The combinations provided by this invention have been evaluated in certain assay systems, and in several different administrative schedules in vitro. The experimental details are as provided herein below. The data presented herein clearly indicate that the two cytotoxic antineoplastic agents, gemcitabine and carboplatin when combined with a CDK inhibitor selected from the compound of formula I exhibit synergistic effect. It is clearly indicated that the anticancer agents when used in combination in the treatment of triple negative breast cancer increases apoptosis or cytotoxicity in proliferative cells than when the cells are treated with only the CDK inhibitor i.e. the compound of formula I alone or the cytotoxic antineoplastic agents alone.

The representative compound, the compound A used in the pharmacological assays refers to (+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl- pyrrolidin-3-yl)-chromen-4-one hydrochloride and was one of the compounds disclosed in the published PCT Publication No. WO2004004632, incorporated herein by reference.

The inventors also established xenograft models to extend in vitro observations to an in vivo system. The inventors tested the combination of the present invention for its in vivo efficacy using triple negative breast cancer xenograft model in SCID mice (Severely Combined Immune Deficient) male mice. It was observed that the CDK inhibitor synergistically enhanced efficacy of the cytotoxic drug combination of gemcitabine and carboplatin when administered in sequential combination with the cytotoxic drug combination.

The synergistic effect of the combination of the present invention comprising two cytotoxic antineoplastic agents, gemcitabine and carboplatin and a CDK inhibitor is now explained in more detail with reference to the embodiments thereof. It is to be noted that these are provided only as examples and not intended to limit the invention.

The following abbreviations or terms are used herein:

ATCC : American Type Culture Collection, USA

ATP : Adenosine triphosphate

CHC1₃ : Chloroform

CDCI₃ : Deuteriated chloroform

CO₂ : Carbon dioxide

CoA : Coenzyme A (Sigma Aldrich, USA)

DCC : N, N'-dicyclohexyl carbodiimide

DBTA : Dibenzoyl tartaric acid

DMAP : 4-Dimethylaminopyridine

DMF : N, N-dimethylformamide

DMSO : Dimethylsulfoxide

DNA : Deoxyribonucleic acid

DTT : Dithiothreitol (Sigma Aldrich, USA)

EDTA : Ethylene diamine tetra acetic acid

EtOAc : Ethyl acetate

FBS : Fetal bovine serum (Gibco, USA)

FCS : Fetal calf serum (Gibco, USA)

g : Gram

h : Hour

HC1 : Hydrochloric acid

IPA : Isopropyl alcohol

KBr : Potassium bromide

Kg : Kilogram L : Litre

MgS0₄ : Magnesium sulfate

MeOH : Methanol

Min : Minute(s)

mL : Millilitre

μL· : Microlitre

μΜ : Micromolar

mmol : Millimolar

mol : Mole

Na₂C0₃ : Sodium carbonate

Na₂S0₄ : Sodium sulfate

NaBH₄ : Sodium borohydride

NaOH : Sodium hydroxide

NCI : National Cancer Institute, USA

°C : Degree Centigrade

PARP : Poly (ADP-ribose) polymerase

PBS : Phosphate buffered saline (Sigma Aldrich, USA)

PI : Propidium iodide (Sigma Aldrich, USA)

RPMI : Roswell Park Memorial Institute, USA

SCID: Severely Combined Immune Deficient, (Jackson Laboratories, USA)

SDS-PAGE : Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

TFA : Trifluoroacetic acid

THF : Tetrahydrofuran Cell-lines (Source: ATCC. USA):

TNBC : Triple negative breast cancer

MCF-7 : (HER low, ER+, PR+, BRCA +/- allelic loss) breast cancer cell-line

T47-D : (HER low, ER +, PR +) breast cancer cell-line

ZR-75-1 : (HER low, ER +, PR +) breast cancer cell-line

MDA-MB-468 : (HER-, ER-, PR-) triple negative breast cancer cell-line

MDA-MB-231 : (HER-, ER-, PR-) triple negative breast cancer cell-line

MDA-MB-435-S : (HER-, ER-, PR-) triple negative breast cancer cell-line

MDA-MB-361 : (HER-, ER-, PR-) triple negative breast cancer cell-line HBL-100 (HER-, ER-, PR-) triple negative breast cancer cell-line

BT-549 (HER-, ER-, PR-) triple negative breast cancer cell-line

HUVEC Human umbilical vein endothelial cells Cell-lines (Source: NCI. USA):

U251 HRE : Genetically engineered glioblastoma cells

U251 pGL3 : Genetically engineered glioblastoma cells

Antibodies (Source: Cell Signaling Technology, USA):

Cyclin Dl (cell cycle protein)

Bcl-2 (anti-apoptotic protein)

CDK4 (cyclin dependent kinase-4)

Rb (Retinoblastoma)

pRb Ser780 (phospho-retinoblastoma)

PAR (substrate of PARP enzyme)

PARP (Poly (ADP-ribose) polymerase)

β-actin (house -keeping protein and used as a loading control for Western blot analysis) Incubation conditions for cell-lines: 37 °C and 5 % CO₂

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

Examples : Example 1:

Preparation of (+)-traras-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxy methyl -1- methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A)

Sodium hydride (50 %, 0.54 g, 11.25 mmol) was added in portions to a solution of (-)-trans- l-[2-Hydroxy-3-(2 -hydro xymethyl-l -methylpyrrolidin-3-yl)-4,6-dimethoxy phenyl)- ethanone (0.7 g., 2.2 mmol) in dry DMF (15 mL) at 0 °C, under nitrogen atmosphere and with stirring. After 10 min., methyl 2-chlorobenzoate (1.15 g., 6.75 mmol) was added. The reaction mixture was stirred at 25 °C for 2 h. Methanol was added carefully below 20 °C. The reaction mixture was poured over crushed ice (300 g), acidified with 1 : 1 HCl (pH 2) and extracted using EtOAc (2 x 100 mL). The aqueous layer was basified using a saturated Na₂CC>3 (pH 10) and extracted using CHCI₃ (3 x 200 mL). The organic layer was dried (anhydrous Na₂S0₄) and concentrated. To the residue, cone. HCl (25 mL) was added and stirred at room temperature for 2 h. The reaction mixture was poured over crushed ice (300 g) and made basic using a saturated aqueous Na₂C0₃ solution. The mixture was extracted using CHCI₃ (3 x 200 mL). The organic extract was washed with water, dried (anhydrous Na₂S0₄) and concentrated to obtain the compound, (+)-ira«s-2-(2-chloro-phenyl)-8-(2- hydroxymethyl- l-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one. [Yield: 0.67 g (64 %); mp: 91- 93°C; [ ]_D ²⁵ = + 5.8° (c = 0.7, methanol)]

Molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added to (+)-trans-2-(2- chloro-phenyl)-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (0.4 g, 0.9 mmol) and heated at 180 °C for 1.5 h. The reaction mixture was cooled to 25 °C, diluted with MeOH (10 mL) and basified using Na₂C0₃ to pH 10. The mixture was filtered and the organic layer was concentrated. The residue was suspended in water (5 mL), stirred for 30 min., filtered and dried to obtain the compound, (+)-ira«s-2-(2-chloro-phenyl)-5,7- dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one. [Yield: 0.25 g (70 %)]

(+)-ira«i-2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)- chromen-4-one (0.2 g, 0.48 mmol) was suspended in IPA (5 mL) and 3.5 % HCl (25 mL) was added. The suspension was heated to get a clear solution. The solution was cooled and solid filtered to obtain the compound, (+)-ira«s-2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2- hydroxymethyl- l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride or Compound A.

Yield: 0.21 g (97 %); mp: 188 - 192 °C ; [ ]_D ²⁵ = +21.3° (c = 0. 2, methanol);

Example 2:

Preparation of (+)-tra«s-2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2- hydroxy- methyl -l-methylpyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound B)

A mixture of the compound of ira«s-l-[2-Hydroxy-3-(2-hydroxymethyl- l- methylpyrrolidin-3-yl)-4,6-dimethoxy phenyl)- ethanone (1.16 g, 3.2 mmol), 2-chloro-4- trifluoromethylbenzoic acid (0.88 g, 4 mmol), DCC (1.35 g, 6.5 mmol) and DMAP (0.4 g, 3.27 mmol) were dissolved in dichloromethane (50 mL) and stirred at room temperature for 12 h. The reaction mixture is cooled to 0 °C, the precipitated dicyclohexylurea was filtered and the organic layer concentrated and residue purified by column chromatography with 1 % methanol in chloroform and 0.01 % ammonia as eluent to obtain the compound, (+)-trans-2- chloro-4-trifluoromethylbenzoic acid 2-(2-acetoxymethyl- 1 -methyl-pyrrolidin-3-yl)-6-acetyl- 3,5-dimethoxyphenyl ester [Yield: 1.44 g (78.8 %)].

To a solution of n-BuLi (15% solution in hexane, 2.2 mL, 5 mmol) in THF (10 mL), maintained at 0 °C under nitrogen atmosphere, hexamethyldisilazane (1.08 mL, 5.1 mmol) was added dropwise and stirred for 15 min. To this, a solution of (+)-ira«s-2-chloro-4- trifluoromethylbenzoic acid 2-(2-acetoxymethyl-l-methyl-pyrrolidin-3-yl)-6-acetyl-3,5- dimethoxyphenyl ester (1.44 g, 2.5 mmol) in THF (10 mL) was added dropwise, maintaining the temperature at 0 °C. After the addition, the reaction was allowed to warm to room temperature and stirred for 2.5 h. The reaction mixture was acidified with dilute HC1, and basified with 10 % sodium bicarbonate to pH 8 to 9. The aqueous layer was extracted with chloroform (3 x 25 mL). The organic layer was washed with water (25 mL), brine (25 mL) and dried over anhydrous Na₂S0₄. The organic layer was concentrated under reduced pressure and dried under vacuum to yield acetic acid 3-{3-[3-(2-chloro-4-trifluromethyl- phenyl)-3-oxo-propionyl] -2-hydroxy-4,6-dimethoxy-phenyl } - l-methyl-pyrrolidin-2-ylmethyl ester as an oil (1.3 g, 90.2 %). This ester was dissolved in cone. HQ (10 mL) and stirred for 3 h to effect cyclisation. At the end of 3 h, the reaction mixture was basified with solid NaHCC>3 to pH 8 to 9. The aqueous layer was extracted with chloroform (25 x 3 mL) and washed with water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous Na₂S0_4, concentrated under reduced pressure and dried over vacuum. The residue was purified by column chromatography with 3 % methanol in chloroform and 0.1 % ammonia as eluent to yield the compound , (+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-8-(2- hydroxymethyl- 1 -methyl pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one as a yellow solid. [Yield: 0.56 g (48.2 %)]

A mixture of (+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-l- methyl pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one (0.25 g, 0.5 mmol), pyridine hydrochloride (0.25 g, 2.16 mmol) and a catalytic amount of quinoline was heated at 180 °C for a period of 2.5 h. The reaction mixture was diluted with methanol (25 mL) and basified with solid Na₂C0₃ to pH 10. The reaction mixture was filtered, and washed with methanol. The organic layer was concentrated and the residue purified by column chromatography using 0.1 % ammonia and 4.5 % methanol in chloroform as eluent to yield the compound, (+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy- methyl-1- methylpyrrolidin-3-yl)-chromen-4-one, as a yellow solid. [Yield: 0.15 g (63.7 %)] (+)-ira«i-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy- methyl- l-methylpyrrolidin-3-yl)-chromen-4-one (0.1 g, 0.2 mmol) was suspended in methanol (2 mL) and treated with ethereal HC1 and the organic solvent evaporated to yield the compound, (+)-ira«i-2-(2-chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l- methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride. [ Yield: O. lg (92.8 %) ]

Pharmacological Assays:

Example 3:

Cytotoxicity assay using Propidium Iodide (PI)

The propidium iodide fluorescence assay (PI) was carried out according to the procedure mentioned in Anticancer Drugs, 1995, 6, 522-532.

The assay was developed to characterize the in vitro growth of human tumor cell lines as well as to test the cytotoxic activity of the compounds. Propidium iodide (PI) was used as a dye, which penetrates only, damaged cellular membranes. Intercalation complexes are formed by PI with double-stranded DNA, which effect an amplification of the fluorescence. After freezing the cells at -20 °C for 24 h, PI had access to total DNA leading to total cell population counts. Background readings were obtained from cell-free wells containing media and propidium iodide.

The human breast cancer cell lines (i.e. MCF-7, T47-D, ZR-75-1, MDA-MB-468,

MDA-MB-231, MDA-MB-435-S, MDA-MB-361, HBL-100, BT-549) were seeded at a density of 1500-3000 cells/well in 180 μΐ. of DMEM (Dulbecco's Modified Eagle's Medium, Gibco, USA) or RPMI 1460, along with 10 % FCS in a 96- well plate and incubated for about 16 h to allow the cells to adhere. The cells were then treated with varying concentrations of the Compound A (0.1 to 3 μΜ). The above procedure was repeated in three TNBC cell-lines (MDA-MB-231, MDA-MB-468 and BT-549) for varying concentrations of Compound A, gemcitabine, carboplatin and BSI-201 (Iniparib developed by Sanofi-Aventis) i.e. the concentration range for Compound A and gemcitabine (Tocris, UK) was 0.01-3 μΜ, while for carboplatin (Shandong Boyuan Chemical Co. Ltd, China) and BSI-201 (prepared in- house) the concentration range was 10-300 μΜ for a total period of 48 h. The plates were incubated in humidified 5 % C0₂ incubator at 37 °C ± 1 °C. Control wells were treated with vehicle (DMSO). At the end of the incubation periods, the plates were assayed using PI cytotoxicity assay protocol. Percent cytoxicity was calculated at various drug concentrations and from the graph plotted the IC5₀ values were determined. The results of this study are presented in Tables 1A and IB.

Table 1A:

Antiproliferative activity of Compound A, BSI-201, Carboplatin and Gemcitabine for TNBC

Table 1A shows that the Compound A when compared with the targeted drugs viz. BSI-201 and Carboplatin showed significantly higher antiproliferative potential. Table IB:

Antiproliferative potential (IC5₀ in μΜ) of Compound A in various breast cancer cell lines as measured by PI assay

Table IB shows that the Compound A was found to be efficaciously antiproliferative against all the breast cancer cell lines irrespective of the genetic markers with IC5₀ ranging from 0.3 to 1.0 μΜ. Example 4:

Clonogenic assay or colony forming assay

MDA-MB-231, MDA-MB-468 and MCF-7 cell lines were seeded in RPMI 1460 with 10 % FCS, at a density of 1500 cells/well in six well plates. After 24 h incubation, cells were treated with ICio, IC30 and IC50 concentrations of Compound A (as determined by the procedure of Example 3) for a period of 48 h and the IC1_0, ICso and IC5₀ values are presented in Table 2. The medium was removed at the end of the treatment and incubated in fresh medium (without drug) for 14 days. After 14 days the medium was aspirated and colonies were fixed with methanol and acetic acid mixture in the proportion of 2: 1, rinsed with water and the fixation procedure was repeated. The plates were dried and colonies stained with 0.1 % crystal violet for 5 min. The wells were finally rinsed with water and dried.

Table 2:

The results are depicted in Figure 1, which shows the visual enhancement in the response by IC₁₀, IC₃₀ and IC₅₀ doses of Compound A, in MDA-MB-231, MDA-MB-468 and MCF-7 cell lines (Seeding density: 1500 cells/plate).

Compound A was found to inhibit the colony forming potential in a dose dependent manner.

Example 5:

Effect of Compound A on Multicellular Tumor Spheroid (3D) formation

The assay was carried out according to the method disclosed in Methods in Molecular Medicine, 2007, 140, 141-151.

The multicellular tumor spheroid (MCTS) model is one of the best-described 3D in vitro tumor model systems, which depicts many of the characteristics of tumor tissue and allows reproducible experiments, offering an excellent in vitro screening system. MCTS were propagated using the hanging drop method. Briefly, the cell monolayer was detached using trypsin-EDTA. Cell count was adjusted and 20 μΐ_^ hanging droplets containing 1,000 cells/drop, were made in bacterial grade petridishes. These hanging drops were incubated for

24 h at 37 °C in a humidified atmosphere of 5 % CO₂. The MCTS thus generated were cultured in the presence or absence of varying concentrations (0.3 μΜ to 30 μΜ) of

Compound A for 72 h.

The results are presented in Figure 2.

When MCF-7 cell suspension was co-incubated with varying concentrations of Compound A (0.3 μΜ to 30 μΜ) for propagation of MCTS, the spheroid formation was arrested from 3 μΜ concentration of Compound A onwards. The size of MCTS formed at 1 μΜ was also smaller as compared to control. This observation is important from the clinical point of view, as MCTS have been characterized sufficiently well to simulate the pathophysiological milieu in a patient tumor. Due to the gradient of oxygen in spheroids, which leads to formation of tumor hypoxia, it mimics the microenvironment prevailing in the tumor tissue. Effect of Compound A on spheroidal formation indicates that Compound A may be effective under hypoxia conditions.

Example 6:

Time dependent effect of Compound A on cell cycle progression and apoptosis in MCF- 7 (Her low, ER+, PR+, BRCA +/- allelic loss) and TNBC cell-line MDA-MB-231

Time dependent effect of Compound A on cell cycle progression and apoptosis was evaluated in two breast cancer cell lines. The asynchronous human breast cancer cell lines MCF-7 (Her low, ER+, PR+, BRCA +/- allelic loss) and MDA-MB-231 cells were seeded in

25 mm³ tissue culture flask at a density of 0.5 xlO⁶ cells per flask in RPMI 1460 with 10 % FCS. After 24 h, cells were treated with 4.5 μΜ of Compound A for 0, 24, 48 and 72 h. Both detached and adherent cells were harvested (trypsinised) at different time points as mentioned in Table 3. After washing in phosphate buffered saline (PBS), cells were fixed in ice-cold 70 % ethanol and stored at -20 °C until further analysis.

Before analysis, cells were washed twice with PBS to remove the fixative and re- suspended in PBS containing 50 μg/mL propidium iodide and 50 μg/mL RNaseA. After incubation at room temperature (20-35 °C) for 20 min, cells were analyzed using flow cytometry. A Becton Dickinson FACS Calibur flow cytometer (BD, USA) was used for these studies. The argon ion laser set at 488 nm was used as an excitation source. Cells with DNA content between 2n and 4n were designated as being in Gl, S and G2/M phases of the cell cycle, as defined by the level of red fluorescence. Cells exhibiting less than 2n DNA content were designated as sub-Gl (apoptotic population) cells. The number of cells in each cell cycle compartment was expressed as a percentage of the total number of cells present. The results are shown in Table 3 and graphically presented in Figure 3A (MCF-7 cell lines) and Figure 3B (MDA-MB-231 cell lines).

Table 3: Percent apoptosis

It is evident from the results shown in the above Table that compound A induced apoptosis in MCF-7 (Her low, ER+, PR+, BRCA +/- allelic loss) and TNBC cell-line MDA-MB-231. Maximum apoptosis was seen at 48h and 72h.

Example 7:

Effect of Compound A in MCF-7 and MDA-MB-231 cells using Western blot analysis:

The Western blot assay was carried out according to the procedure disclosed in

Molecular Cancer Therapeutics, 2007, 6, 918-925 with some modifications.

MCF-7 and MDA-MB-231 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm³ tissue culture flask and incubated for 24 h. The cells were treated with Compound A at 1.5 and 4.5 μΜ. At various time points, i.e. 6, 24 and 30 h the cells were harvested or trypsinized and lysed using lysis buffer (Sigma Aldrich, USA). Protein content was estimated. Lysate was applied to Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by western blotting (Molecular Cancer Therapeutics, 2007, 6, 918-925). Western blotting was done using specific antibodies to Bcl-2 and actin. The results are depicted in Figure 4.

It can be seen from Figure 4 that compound A down regulates antiapoptotic protein

Bcl-2 in a dose dependent manner in both the cell lines. In MCF-7 cells, Bcl-2 is significantly down regulated from 24 h onwards, while in MDA-MB-231 significant down regulation was observed at 30 h. Example 8:

Effect of Compound A on cell cycle progression and apoptosis:

Comparison of the effect of Compound A and PARP inhibitor BSI-201 (prepared in- house) on cell cycle progression and apoptosis was evaluated in two TNBC cell lines. The asynchronous human TNBC cell lines MDA-MB-231 and MDA-MB-468 were seeded in 25 mm³ tissue culture flask at a density of 0.5 xlO⁶ cells per flask in RPMI 1460 with 10 % FCS. After 24 h, cells were treated either with 1.5 and 3.0 μΜ of Compound A or 50 μΜ of PARP inhibitor BSI-201 for 72 h. After the incubation cells were harvested (trypsinised) and processed as given in example 6. The results are shown in Tables 4A and 4B; and graphically presented in Figures 5A, 5B and 5C.

Table 4A: Comparative analysis of percentage distribution of cells in different cell cycle phases and apoptosis in MDA-MB-231 treated with Compound A (a CDK inhibitor) and BSI-201 (a PARP inhibitor)

Table 4B: Comparative analysis of percentage distribution of cells in different cell cycle phases and apoptosis in MDA-MB-468 treated with Compound A (a CDK inhibitor) and BSI-201 (a PARP inhibitor)

The TNBC cell lines MDA-MB-231 and MDA-MB-468 showed dose dependent increase in apoptosis when treated with Compound A. BSI-201 (at 50 μΜ) showed no induction of apoptosis in MDA-MB-231. However, marginal apoptosis (12.67 %) was observed in MDA-MB-468.

Example 9:

Effect of Compound A on MCF-7 cell cycle proteins and CDK4 kinase activity

Step 1: Basal level of cyclin-Dl expression

Basal level of cyclin-Dl expression was studied using western blot analysis (Molecular Cancer Therapeutics, 2007, 6, 918-925) across various breast cancer cell lines, i.e. MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-435 S, MDA-MB-453, BT-549 and HBL-100. These cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm³ tissue culture flask and incubated for 24 h. The cells were harvested (trypsinised) and lysed using lysis buffer. Protein content was estimated. Lysate was applied to Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by Western blotting. Western blotting was done using cyclin Dl antibody and actin is used as a loading control. The results are shown in Figure 6A. High cyclin Dl levels were observed in most of the breast cancer cell lines including triple negative breast cancer cell lines.

Step 2: Effect of Compound A on MCF-7 cell cycle proteins and CDK4 kinase activity

MCF-7 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm³ tissue culture flask and incubated for 24 h. These cells were treated with Compound A at 1.5 μΜ. At various time points viz. 3 h, 6 h, 9 h, 12 h and 24 h the cells were harvested (trypsinised) and lysed using lysis buffer. Protein content was estimated by Bradford method (Anal. Biochem., 1976, 72, 248-254). Lysate was applied to Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by Western blotting. Western blotting was done using specific antibodies to various cell cycle proteins viz. cyclin Dl, CDK4, Rb and pRbSer780.

For immunoprecipitation assay, the MCF-7 cells were synchronized by serum starvation. These cells were treated with Compound A at 1.5 μΜ at various time points viz. 3 h, 6 h, 9 h and 12 h. Cells were harvested (trypsinised) and lysed using lysis buffer, and protein content was estimated. CDK4-D1 (Cyclin Dl and CDK4) was purified from the lysate by immunoprecipitation using specific antibody to CDK4. Immune complex was further purified using Protein A sepharose beads (Sigma Aldrich, USA). Immune complex was used to determine CDK4 activity using pRb as a substrate and ³²P labelled ATP (BRIT, India). Reaction mixed was applied to SDS-PAGE followed by transfer and autoradiography. The results are shown in Figure 6B.

Compound A down regulates cyclin Dl and pRb in MCF-7 (Her low, ER+, PR+, BRCA +/- with allelic loss) in time dependent manner. Cyclin Dl and pRb expression show decrease from 6 h onwards and significantly at 12 h. There is no significant change in total Rb except at 24 h. Decrease in CDK4 kinase activity in cell-based assay was seen as early as 3 h onwards.

Example 10:

Effect of Compound A on PARP enzyme activity as measured by PAR polymers

Poly(ADP-ribose) polymerase (PARP) is the principle member of a family of enzyme possessing poly(ADP-ribosylation) (PAR) catalytic capacity. To study PARP enzyme activity, PAR polymer formation was measured. MDA-MB-231 and MDA-MB-468 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm³ tissue culture flask and incubated for 24 h. These cells were treated at 1.5 μΜ and 5 μΜ of Compound A for 24 h. The cells were harvested (trypsinised) and lysed using lysis buffer. Western blotting (Molecular Cancer Therapeutics, 2007, 6, 918-925) was done with specific antibody to PAR. The results are shown in Figure 7.

Compound A inhibits PARP enzyme activity as observed by the inhibition of PAR polymer formation in MDA-MB-231 cell line. However it was observed that in MDA-MB- 468 the formation of PAR polymers is not inhibited.

Example 11:

Effect of Compound A (24 h) on PARP and cell cycle proteins in TNBC cell lines

Correlation of PARP activity and cell cycle proteins cyclin Dl, total Rb and pRb 780 were studied in two TNBC cell lines viz. MDA-MB-468 and MDA-MB-231. MDA-MB-231 and MDA-MB-468 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm³ tissue culture flask and incubated for 24 h. These cells were treated at 1.5 μΜ and 5 μΜ Compound A for 24 h. The cells were harvested (trypsinised) and lysed using lysis buffer. Western blotting was carried out (Molecular Cancer Therapeutics, 2007, 6, 918-925) using specific antibody to PAR, PARP, cyclin Dl, CDK4 and pRb Ser 780. The results are shown in Figure 8.

In MDA-MB-231, Compound A inhibits PARP enzyme activity as seen by the inhibition of PAR polymer formation. This is accompanied by dose dependent decrease in pRb, cyclin Dl and CDK4. While in MDA-MB-468 although there was no change in the PAR polymer formation, PARP cleavage was prominent, which is an indication of apoptosis.

On treatment of TNBC cell line MDA-MB-231 with the compound A and incubation for 24h, inhibition of PARP activity was observed in the cell line. However, MDA-MB-468 did not show PARP enzyme inhibition and instead showed cleaved PARP. Both of these are markers of cells undergoing apoptosis. Thus, it is evident that compound A induces significant apoptosis in both these cell lines.

Example 12:

Effect of Compound A on HIF-lct inhibition

Test System in HIF-Ια reporter gene based assay:

1) U251 HRE: The genetically engineered cells U251 HRE which stably express a recombinant vector in which the Luciferase reporter gene is under control of three copies of a canonical HRE

2) U251 pGL3: A control cell line contains the firefly Luciferase reporter gene under control of the constitutively active SV40 promoter and enhancer that helps to exclude compounds that inhibit Luciferase expression in a nonspecific and/or HIF-1 -independent fashion. These cells expressed high basal levels of Luciferase in normoxic conditions and slightly lower levels in hypoxic conditions.

U251 HRE cells were inoculated into 96 well white flat-bottomed plates at 10000 -

15000 cells/well in a volume of 180 and incubated for 24 h at 37 °C, 5 % C0₂, and ambient 0₂. Compound A was tested at various concentrations viz. 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10 μΜ and plates were incubated for 20 h in a modular hypoxia chamber (Billups Rothenberg, MIC 101, USA) at 37 °C, 5 % C0₂, 1 % 0₂ and 94 % N₂. After 20 h incubation, the plates were removed and incubated at room temperature, 5 % C0₂, and ambient 0₂ for 1.5 h. 40 of Bright Glo Luciferase reagent (Promega , USA) was added, and after 3 min, luminescence was measured using a Polar Star Plate Reader (USA) in luminescence mode. Appropriate control cells (U251 pGL3) were treated identically, except that they were treated at 37 °C, 5 % C0₂, and ambient 0_2. Compound toxicity was assayed using the MTS assay.

Treatment with Compound A effectively blocked the expression of HIF-1 a in a dose dependent manner in the U251 HRE cell line under hypoxia (< 1 % 0₂). These compounds did not inhibit the luciferase expression in the control cell line U251 pGL3 under normoxia. This indicates that the Compound A inhibits HIF-1 a specifically. The results are graphically presented in Figure 9.

Example 13:

Effect of Compound A on VEGF inhibition:

A cell line M-9 is MDA-MB-231 which is stably co-transfected with the VEGF- Luc construct (VEGF promoter in pGL2-basic) and a plasmid containing the Geneticin (G418) resistance gene which forms VEGF promoter reporter gene. The expression of the reporter gene in the clone cells, as measured by luciferase activity, is stable.

The effect of Compound A on VEGF inhibition was evaluated using the VEGF reporter gene based assay.

Reagents for VEGF assay:

Lysis Assay Buffer (IX)

Tris-Phosphate (pH 7.8)-125 mM, DTT-10 mM, EDTA-10 mM, Glycerol-50 % and Triton X- 100-5 %.

Luciferase Assay Reagent (LAR)

Luciferase Assay Buffer-8 mL, 530 μΜ ATP-530 μΐ,, 270 μΜ CoA-1 mL and 170 μΜ Luciferin-1 mL.

Luciferase Assay Buffer (LAB) (IX)

Tricin (pH 7.8)-20 mM, Magnesia Alba-1.07 mM, MgS04-2.67 mM, EDTA-0.1 mM and DTT-33.3 mM.

ATP Stock made in LAB = 5.85 mg/mL

CoA Stock made in LAB = 2.1 mg/mL

Luciferin Stock made in LAB = 1.5 mg/mL

Protocol for VEGF assay:

1. M-9 cells were sub-cultured and maintained in RPMI-1640 Medium with supplement of 10 % FBS, and 4 μίΛηΙ G418 (Stock 100 mg/mL) in a humidified incubator at 37 °C and 5 % C0₂.

2. Cells were seeded at density of 3 x 10⁴ cells/well in 180 μL· volume in tissue culture grade 96 well white plates as well as transparent plates and allowed to adhere for 16-20 h in humidified CO₂ incubator (5 % CO₂) at 37 °C. A total of two sets of plates were made, as the incubation conditions are different.

3. Compound A and BSI-201 were diluted in medium serially so that final desired concentrations are achieved in the respective wells, (not more than 0.5 % concentration of DMSO in the wells). 4. INCUBATION CONDITIONS: One set of plates are incubated under ambient atmospheric condition with 5 % C0₂, referred hereafter as NORMOXIC/OXIC PLATE. While the other set of plate goes in anoxic condition where the Oxygen concentration is less the 1 %, and 94 % Nitrogen, 5 % CO₂, and referred hereafter as HYPOXIC PLATE. Temperature of incubation is 37 °C and humidity greater than 75 %.

5. After 20-24 h incubation under Hypoxic and Normoxic conditions, plates are taken out from the incubators, medium from all the wells are removed from white plate. Cells are given a rapid wash with 100-150 μίΛνεΙΙ Phosphate Buffer Saline (PBS). Cells are lysed with 40- 50 μΕ Lysis buffer for 20 min.

6. To all the wells, 100 Luciferase Assay Reagent (LAR) are added, plates are immediately read for Luminescence on TOPCOUNT™ (Packard, USA). The percentage inhibitions and Inhibitory Concentration (IC5₀) or Effective Concentration (EC5₀) are calculated in comparison with Control (untreated) values. IC5₀ values (μΜ) for VEGF inhibition under hypoxia:

Compound A : 0.31 μΜ

BSI-201 : > 100 μΜ

The results are graphically presented in Figure 10.

Treatment with Compound A effectively blocked the expression of VEGF in a dose dependent manner.

Example 14:

Effect of Compound A in wound healing assay:

The wound-healing assay is simple, inexpensive, and one of the earliest developed methods to study directional cell migration in vitro. This method mimics cell migration during wound healing in vivo.

Protocol:

1. MCF-7 cells were seeded in RPMI 1460 medium with 10 % FCS in 25 mm³ tissue culture flask and incubated for 24 h

2. The cells were trypsinized and seeded at a density of (0.5 - 2.0) x 10⁶ per well in a sterile 6 well plate.

3. The plate was incubated for about 16 h in humidified CO₂ incubator (5 % CO₂) at 37° C under ambient oxygen levels. The cells were observed to form a confluent uniform monolayer on the complete surface of the well. The required number of cells for a confluent monolayer depends on both the particular cell type and size of dishes.

4. The cell monolayer in a straight line was scraped evenly to create a "scratch" with a pipette tip. The first image of the scratches was captured before addition of compound.

5. Compound A was added at concentrations of 1 μΜ and 3 μΜ.

6. The plates were then kept in the incubator for further incubation. The time frame for incubation was determined empirically for the particular cell type used.

7. After the incubation, the dish was placed under a phase contrast microscope (Zeiss Axio Observer, Germany), reference point was matched, the photographed regions of the first image were aligned and the second image was captured. For each image distances between one side of the scratch and the other were measured.

Similar protocol was followed for BT-549 and MDA-MB-231 cell lines.

The results are presented in Figures 11A, 11B and 11C.

Compound A showed potent anti migratory effect in all the breast cancer cell lines including triple negative breast cancers cell lines. The control cells showed complete healing after an incubation of 24 h. The cells treated with Compound A showed very less migration from both sides, thus indicating potent anti migratory effect.

Example 15:

Angiogenesis of Compound A in Endothelial Tube Formation Assay

The Tube Formation Assay represents a simple but powerful model for studying inhibition and induction of angiogenesis. The assay relies on the endothelial cells' ability to form distinct blood-vessel like tubules in an extracellular matrix (BD Matrigel™ Matrix, USA) where they can subsequently be visualized by microscopy. It enables analysis of angiogenic tubules in a 3 dimensional matrix that better resembles the native physiological environment.

Protocol

Endothelial Cell Tube Formation Assay

Confluent HUVEC (Human umbilical vein endothelial cells) were cultured with above mentioned endothelial medium to desired confluence. For HUVEC 60-80 % confluence is recommended.

Endothelial cell suspensions were prepared by trypsinizing the cell monolayers and resuspending the cells in culture medium with 5-10 % serum. (0.5 - l) x l0⁶ cells per 180 μΕ of cell suspension were added (per well of 24 well plate) to the medium (BD Matrigel Matrix) which, had been thawed at 4°C. This suspension was then added to the plates and kept for incubation. The cells were allowed to adhere for 2-3 h and then Compound Α(1μΜ), Rotenone (l uM) (Sigma- Aldrich, USA) and Topotecan (3μΜ) (Sigma- Aldrich, USA) (20 μΐ. of 10X stocks) were added to the respective wells. DMSO was used as the control. After 24 - 48 h of incubation the cells were observed under a phase contrast microscope (Zeiss Axio Observer, Germany) for tube formation and angiogenesis.

The results are shown in Figure 12.

Compound A effectively inhibited endothelial tube formation and thus angiogenesis in the 3D gel HUVEC tube formation assay. Compound A at 1 μΜ was comparable to Rotenone (standard VEGF inhibitor) and better than Topotecan (known HIF-Ι inhibitor in clinical trials).

Example 16:

In vitro cytotoxicity assay:

Methods

Assay protocol for Propidium Iodide (PI) assay

The propidium iodide fluorescence assay (PI) was carried out according to the procedure mentioned in Anticancer Drugs, 1995, 6, 522-32.

The assay was developed to characterize the in vitro growth of human tumor cell lines as well as to test the cytotoxic activity of the test compounds. Propidium iodide (PI) was used as a dye, which penetrates only, damaged cellular membranes. Intercalation complexes are formed by PI with double-stranded DNA, which effect an amplification of the fluorescence. After freezing the cells at -20 °C for 24 h, PI had access to total DNA leading to total cell population counts. Background readings were obtained from cell-free wells containing media and propidium iodide.

The human triple negative breast cancer cell line, MDA-MB-231 was seeded at a density of 1500-3000 cells/well in 180 μΐ. of RPMI-1640 medium in a 96-well plate and incubated for about 16 h in humidified 5 % CO₂ incubator at 37 ± 1 °C to allow the cells to adhere. The cells were then treated with two different schedules. In every schedule, 20 μυ of 10X compound (dissolved first in DMSO and then diluted in cell medium, final DMSO concentration not exceeding 0.5 %) was used in the wells and the plate was incubated in humidified 5 % CO₂ incubator at 37 ± 1 °C. The medium was removed from the wells and washed with PBS. 100 μυ of PI working solution (7 μg/mL) per well was added and the plates were stored at -80 °C for about 16 h. The plates were thawed and the fluorescence was measured using the POLARstar optima plate reader (USA) at excitation 536 nm and emission 590 nm.

(PI stock solution of lmg/mL was prepared by dissolving 1 mg PI in 1 mL of distilled water. PI working solution was prepared by adding 140 μί of stock solution to PBS to make up the volume to 220 mL (7 μg/mL)).

Regimen 1:

Part A: It consists of 4 treatment groups.

1) The MDA-MB-231 cells were treated with DMSO vehicle and incubated for 24 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 72 h (Group IA).

2) The cells were treated with complete medium and incubated for 24 h followed by removal of medium, addition of Compound A (ICso=l μΜ) and incubation for 72 h. (Group IIA).

3) The cells were treated with Gemcitabine (IC₃o=0.46 μΜ) and Carboplatin (IC₃o=12.0 μΜ) together and incubated for 24 h followed by removal of medium, addition of complete medium and incubation for 72 h (Group IIIA). (Serial double dilutions for Gemcitabine and Carboplatin were carried out for a total of 6 concentrations and % inhibition was measured).

4) The cells were treated with Gemcitabine (IC₃₀) and Carboplatin (IC₃₀) together and incubated for 24 h followed by removal of medium, addition of Compound A and incubation for 72 h (Group IVA). (Serial double dilutions for Gemcitabine and Carboplatin were carried out for a total of 6 concentrations and % inhibition was measured).

The schedule of drug treatment is shown in Table 5A.

Table 5A. Triple drug combination regimen 1 for 72 h

Group Schedule for drug treatment

At O h At 24 h

IA Vehicle control and 24 h incubation CM and 72 h incubation

IIA CM and 24 h incubation Compound A (ICso=l μΜ) and 72 h incubation

IIIA Gemcitabine (IC₃o=0.46 μΜ) and Carboplatin CM and 72 h (IC₃₀=12.0 μΜ) and 24 h incubation incubation

Gemcitabine (0.23 μΜ) and Carboplatin (6.0 μΜ)

and 24 h incubation

Gemcitabine (0.115 μΜ) and Carboplatin (3.0

μΜ) and 24 h incubation

Gemcitabine (0.06 μΜ) and Carboplatin (1.5 μΜ)

and 24 h incubation

Gemcitabine (0.03 μΜ) and Carboplatin (0.75

μΜ) and 24 h incubation

Gemcitabine (0.015 μΜ) and Carboplatin (0.375

μΜ) and 24 h incubation

IVA Gemcitabine (IC₃o=0.46 μΜ) and Carboplatin Compound A (ICso=l (IC₃₀=12.0 μΜ) and 24 h incubation μΜ) and 72 h

Gemcitabine (0.23 μΜ) and Carboplatin (6.0 μΜ) incubation

and 24 h incubation

Gemcitabine (0.115 μΜ) and Carboplatin (3.0

μΜ) and 24 h incubation

Gemcitabine (0.06 μΜ) and Carboplatin (1.5 μΜ)

and 24 h incubation

Gemcitabine (0.03 μΜ) and Carboplatin (0.75

μΜ) and 24 h incubation

Gemcitabine (0.015 μΜ) and Carboplatin (0.375

μΜ) and 24 h incubation

CM: complete medium

Part B: It consists of 4 treatment groups.

1) The cells were treated with DMSO vehicle and incubated for 24 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 96 h (Group IB). 2) The cells were treated with complete medium and incubated for 24 h followed by removal of medium, addition of Compound A (IC5₀) and incubation for 96 h (Group IIB).

3) The cells were treated with Gemcitabine (IC₃₀) and Carboplatin (IC₃₀) together and incubated for 24 h followed by removal of medium, addition of complete medium and incubation for 96 h (Group IIIB). (Serial double dilutions for Gemcitabine and Carboplatin were carried out for a total of 6 concentrations and % inhibition was measured).

4) The cells were treated with Gemcitabine (IC₃₀) and Carboplatin (IC₃₀) together and incubated for 24 h followed by removal of medium, addition of Compound A and incubation for 96 h (Group IVB). (Serial double dilutions for Gemcitabine and Carboplatin were carried out for a total of 6 concentrations and % inhibition was measured).

The schedule of drug treatment (triple drug combination regimen 1) is shown in Table 5B.

Table 5B. Triple drug combination regimen 1 for 96 h

Group Schedule for drug treatment

At O h At 24 h

IB Vehicle control and 24 h incubation CM for 96 h

IIB CM and 24 h incubation Compound A

μΜ) for 96 h

IIIB Gemcitabine (IC₃o=0.46 μΜ) and Carboplatin CM and 96 h incubation

(IC₃₀=12.0 μΜ) and 24 h incubation

Gemcitabine (0.23 μΜ) and Carboplatin (6.0

μΜ) and 24 h incubation

Gemcitabine (0.115 μΜ) and Carboplatin (3.0

μΜ) and 24 h incubation

Gemcitabine (0.06 μΜ) and Carboplatin (1.5

μΜ) and 24 h incubation

Gemcitabine (0.03 μΜ) and Carboplatin (0.75

μΜ) and 24 h incubation

Gemcitabine (0.015 μΜ) and Carboplatin

(0.375 μΜ) and 24 h incubation IVB Gemcitabine (IC₃o=0.46 μΜ) and Carboplatin Compound A (ICso=l (IC₃₀=12.0 μΜ) and 24 h incubation μΜ) and 96 h

Gemcitabine (0.23 μΜ) and Carboplatin (6.0 incubation

μΜ) and 24 h incubation

Gemcitabine (0.115 μΜ) and Carboplatin (3.0

μΜ) and 24 h incubation

Gemcitabine (0.06 μΜ) and Carboplatin (1.5

μΜ) and 24 h incubation

Gemcitabine (0.03 μΜ) and Carboplatin (0.75

μΜ) and 24 h incubation

Gemcitabine (0.015 μΜ) and Carboplatin

(0.375 μΜ) and 24 h incubation

The results for Regimen 1 (Part A and Part B) are shown in Figures 13A and 13B.

Figures 13A and 13B shows the efficacy of the combination of various concentrations of Gemcitabine and Carboplatin together for 24 h followed by Compound A for 72 and 96 h in MDA-MB-231 cell line. The combination of Gemcitabine and Carboplatin together for 24 h followed by IC5₀ of Compound A (1.0 μΜ) for 72 and 96 h was found to be synergistic in MDA-MB-231 cell line.

Cytotoxicity determination:

The IC5₀ values in μΜ for BSI-201 , Carboplatin, Gemcitabine, and Compound A in

MDA-MB-231 , BT-549 and MDA-MB-468 determined by cytotoxicity assay done after 48 h of compound treatment as determined in Table 1A of Example 3 were used in Example 16. After completion of the compound treatment i.e. at the end of 48 h, the plates were processed for PI assay and the percent cytotoxicity was calculated as compared to DMSO (vehicle) control. The results indicate that BSI-201, a PARP inhibitor shows seventy times higher IC5₀ as compared to Compound A in all TNBC cell lines. Compound A shows greater potency in TNBC cell lines as compared to Carboplatin and BSI-201 in antiproliferative assay. Regimen 2A: In each of Parts A, B, C and D, 4 groups were set-up.

Part A:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 72 h (Group la).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of Compound A (¾ο=0.75 μΜ) and incubation for 72 h (Group 2a).

3) The cells were treated with Gemcitabine (ICio=0.3 μΜ) and incubated for 6 h followed by treatment with Carboplatin (ICio=6.0 μΜ) and incubation for 24 h followed further by removal of medium, addition of complete medium and incubation for 72 h (Group 3a).

4) The cells were treated with Gemcitabine (IC₁₀) and incubated for 6 h followed by treatment with Carboplatin (IC_lo) and incubation for 24 h followed further by removal of medium, addition of Compound A (IC₃₀) and incubation for 72 h (Group 4a).

The schedule of drug treatment (triple drug combination regimen 2) is shown in Table 6A.

Table 6A. Triple drug combination regimen 2 with Compound A (IC₃o=0.75 μΜ) and incubation for 72 h

Group Schedule for drug treatment

At O h At 30 h

la Vehicle control and 30 h incubation CM and 72 h incubation

2a CM and 30 h incubation Compound A (IC₃₀=0.75 μΜ) and

72 h incubation

At O h At 6 h At 30 h

3a Gemcitabine Carboplatin CM and 72 h incubation

(ICio=0.3 μΜ) (ICio=6.0 μΜ)

and 6 h and 24 h

incubation incubation

4a Gemcitabine Carboplatin Compound A (IC₃₀=0.75 μΜ) and

(ICio=0.3 μΜ) (ICio=6.0 μΜ) 72 h incubation

and 6 h and 24 h

incubation incubation Part B:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 96 h (Group lb).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of Compound A (IC₃₀) and incubation for 96 h (Group 2b).

3) The cells were treated with Gemcitabine (IC₁₀) and incubated for 6 h followed by treatment with Carboplatin (IC_lo) and incubation for 24 h followed further by removal of medium, addition of complete medium and incubation for 96 h (Group 3b).

4) The cells were treated with Gemcitabine (IC_lo) and incubated for 6 h followed by treatment with Carboplatin (IC_lo) and incubation for 24 h followed further by removal of medium, addition of Compound A (IC₃o=0.75 μΜ) and incubation for 96 h (Group 4b).

The schedule of drug treatment (triple drug combination regimen 2) is shown in Table 6B. Table 6B. Triple drug combination regimen 2 with Compound A (IC₃o=0.75 μΜ) for 96 h

Group Schedule for drug treatment

At O h At 30 h

lb Vehicle control and 30 h incubation CM and 96 h incubation

2b CM and 30 h incubation Compound A (IC₃₀=0.75 μΜ) and

96 h incubation

At O h At 6 h At 30 h

3b Gemcitabine Carboplatin CM and 96 h incubation

(ICio=0.3 μΜ) (ICio=6.0 μΜ)

and 6 h and 24 h

incubation incubation

4b Gemcitabine Carboplatin Compound A (IC₃₀=0.75 μΜ) and

(ICio=0.3 μΜ) (ICio=6.0 μΜ) 96 h incubation

and 6 h and 24 h

incubation incubation Part C:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 72 h (Group la).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of Compound A (IC5₀) and incubation for 72 h (Group 2c).

3) The cells were treated with Gemcitabine (IC₁₀) and incubated for 6 h followed by treatment with Carboplatin (IC_lo) and incubation for 24 h followed further by removal of medium, addition of complete medium and incubation for 72 h (Group 3a).

4) The cells were treated with Gemcitabine (IC_lo) and incubated for 6 h followed by treatment with Carboplatin (IC_lo) and incubation for 24 h followed further by removal of medium, addition of Compound A (IC5₀) and incubation for 72 h (Group 4c).

The schedule of drug treatment (triple drug combination regimen 2) is shown in Table 6C. Table 6C. Triple drug combination regimen 2 with Compound A (ICso=l -0 μΜ) for 72 h

Group Schedule for drug treatment

At O h At 30 h

la Vehicle control and 30 h incubation CM and 72 h incubation

2c CM and 30 h incubation Compound A (IC₅₀=1.0 μΜ) and

72 h incubation

At O h At 6 h At 30 h

3a Gemcitabine Carboplatin CM and 72 h incubation

(ICio=0.3 μΜ) (ICio=6.0 μΜ)

and 6 h and 24 h

incubation incubation

4c Gemcitabine Carboplatin Compound A (IC₅₀=1.0 μΜ) and

(ICio=0.3 μΜ) (ICio=6.0 μΜ) 72 h incubation

and 6 h and 24 h

incubation incubation Part D:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 96 h (Group lb).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of Compound A (IC₅₀) and incubation for 96 h (Group 2d).

3) The cells were treated with Gemcitabine (ICio) and incubated for 6 h followed by treatment with Carboplatin (ICio) and incubation for 24 h followed by removal of medium, addition of complete medium and incubation for 96 h (Group 3b).

4) The cells were treated with Gemcitabine (ICio) and incubated for 6 h followed by treatment with Carboplatin (ICio) and incubation for 24 h followed further by removal of medium, addition of Compound A (IC₅₀) and incubation for 96 h (Group 4d).

The schedule of drug treatment (triple drug combination regimen 2) is shown in Table 6D. Table 6D. Triple drug combination regimen 2 with Compound A (ICso=l -0 μΜ) for 96 h

For the above experiments, at the end of the incubation periods, the plates were assayed using the PI cytotoxicity assay protocol. The results are indicated in Figures 14A and 14B. Figures 14A and 14B shows the efficacy of the combination of Gemcitabine and Carboplatin with Compound A for 72 and 96 h in MDA-MB-231 cell line. At lower concentrations, ICio of Gemcitabine and Carboplatin, the percent cytotoxicity is 49 % and 63.9 % at the end of treatment period of 96 h and 120 h respectively. However, Compound A at both the IC₃₀ and IC5₀ is able to significantly potentiate the cytotoxicity caused by Gemcitabine and Carboplatin as compared to Gemcitabine and Carboplatin alone to 60 % and 74.5 % respectively at 72 h. and similarly 77.7 % and 88.2 % respectively at 96 h. Hence longer the period of Compound A treatment (96 h) and higher the concentration of Compound A (i.e. IC5₀ concentration) greater is the cytotoxicity.

Regimen 2B: Combination studies with BSI-201 and comparison with combination studies of Compound A in MDA-MB-231 cells

In each of Parts A, B, C and D, 4 groups were set-up.

Part A:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 72 h (Group la*).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of BSI-201 (IC₃₀) and incubation for 72 h (Group 2a*).

3) The cells were treated with Gemcitabine (ICio) and incubated for 6 h followed by treatment with Carboplatin (ICio) and incubation for 24 h followed by removal of medium, addition of complete medium and incubation for 72 h (Group 3a*).

4) The cells were treated with Gemcitabine (IC₁₀) and incubated for 6 h followed by treatment with Carboplatin (IC_lo) and incubation for 24 h followed by removal of medium, addition of BSI-201 (IC₃₀) and incubation for 72 h (Group 4a*). The schedule of drug treatment is shown in Table 6A*.

Table 6A*: Triple drug combination regimen 2 with BSI-201 (IC₃o=40 μΜ) for 72 h

Group Schedule for drug treatment

At O h At 30 h

la* Vehicle control and 30 h incubation CM and 72 h incubation

2a* CM and 30 h incubation BSI-201 (IC₃₀=40 μΜ) and 72 h

incubation At O h At 6 h At 30 h

3a* Gemcitabine Carboplatin CM and 72 h incubation

(ICio=0.3 μΜ) (ICio=6.0 μΜ)

and 6 h and 24 h

incubation incubation

4a* Gemcitabine Carboplatin BSI-201 (IC₃₀=40 μΜ) and 72 h

(ICio=0.3 μΜ) (ICio=6.0 μΜ) incubation

and 6 h and 24 h

incubation incubation

Part B:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 96 h (Group lb*).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium addition of BSI-201 (IC30) and incubation for 96 h (Group 2b*).

3) The cells were treated with Gemcitabine (IC1₀) and incubated for 6 h followed by treatment with Carboplatin (IC1₀) and incubation for 24 h followed further by removal of medium, addition of complete medium and incubation for 96 h (Group 3b*).

4) The cells were treated with Gemcitabine (IC1₀) and incubated for 6 h followed by treatment with Carboplatin (IC1₀) and incubation for 24 h followed further by removal of medium, addition of BSI-201 (IC₃₀) and incubation for 96 h (Group 4b*).

The schedule of drug treatment is shown in Table 6B*.

Table 6B*: Triple drug combination regimen 2 with BSI-201 (IC₃o=40 μΜ) for 96 h

Group Schedule for drug treatment

At O h At 30 h

lb* Vehicle control and 30 h incubation CM and 96 h incubation

2b* CM and 30 h incubation BSI-201 (IC₃₀=40 μΜ) and 96 h

incubation

At O h At 6 h At 30 h

3b* Gemcitabine Carboplatin CM and 96 h incubation

(ICio=0.3 μΜ) (ICio=6.0 μΜ) and 6 h and 24 h

incubation incubation

4b* Gemcitabine Carboplatin BSI-201 (IC₃₀=40 μΜ) and 96 h

(ICio=0.3 μΜ) (ICio=6.0 μΜ) incubation

and 6 h and 24 h

incubation incubation

Part C:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 72 h (Group la*).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of BSI-201 (IC5₀) and incubation for 72 h (Group 2c*).

3) The cells were treated with Gemcitabine (IC1₀) and incubated for 6 h followed by treatment with Carboplatin (IC1₀) and incubation for 24 h followed further by removal of medium, addition of complete medium and incubation for 72 h (Group 3a*).

4) The cells were treated with Gemcitabine (IC1₀) and incubated for 6 h followed by treatment with Carboplatin (IC1₀) and incubation for 24 h followed further by removal of medium, addition of BSI-201 (IC5₀) and incubation for 72 h (Group 4c*).

The schedule of drug treatment is shown in Table 6C*.

Table 6C*: Triple drug combination regimen 2 with BSI-201 (ICso=70 μΜ) for 72 h

Group Schedule for drug treatment

At O h At 30 h

la* Vehicle control and 30 h incubation CM and 72 h incubation

2c* CM and 30 h incubation BSI-201 (IC₅₀=70 μΜ) and

72 h incubation

At O h At 6 h At 30 h

3a* Gemcitabine Carboplatin CM and 72 h incubation

(ICio=0.3 μΜ) and 6 (ICio=6.0 μΜ) and

h incubation 24 h incubation

4c* Gemcitabine Carboplatin BSI-201 (IC₅₀=70 μΜ) and

(ICio=0.3 μΜ) and 6 (ICio=6.0 μΜ) and 72 h incubation

h incubation 24 h incubation Part D:

1) The cells were treated with DMSO vehicle and incubated for 30 h followed by removal of medium, addition of complete medium (CM: medium + 10 % FCS) and incubation for 96 h (Group lb*).

2) The cells were treated with complete medium and incubated for 30 h followed by removal of medium, addition of BSI-201 (IC₅₀) and incubation for 96 h (Group 2d*).

3) The cells were treated with Gemcitabine (ICio) and incubated for 6 h followed by treatment with Carboplatin (ICio) and incubation for 24 h followed further by removal of medium, addition of complete medium and incubation for 96 h (Group 3b*).

4) The cells were treated with Gemcitabine (ICio) and incubated for 6 h followed by treatment with Carboplatin (ICio) and incubation for 24 h followed further by removal of medium, addition of BSI-201 (IC₅₀) and incubation for 96 h (Group 4d*).

The schedule of drug treatment is shown in Table 6D*. Table 6D*: Triple drug combination regimen 2 with BSI-201 (ICso=70 μΜ) for 96 h

For the above experiments, at the end of the incubation periods, the plates were assayed using the PI cytotoxicity assay protocol. The results are indicated in Figures 15A and 15B. Figures 15A and 15B show the efficacy of the combination of Gemcitabine and Carboplatin with BSI-201 for 72 and 96 h in MDA-MB-231 cell line. At lower concentrations, i.e. ICio of Gemcitabine and Carboplatin, the percent cytotoxicity is 59.7 % and 49.9 % at the end of treatment period of 96 h and 120 h respectively.

However, BSI-201 as compared to Compound A at both IC30 and IC50 is not able to significantly potentiate the cytotoxicity caused by Gemcitabine and Carboplatin as compared to Gemcitabine and Carboplatin alone that is only 62.9 % and 63.2 % respectively at 72 h. and similarly 47.1 % and 57.3 % respectively at 96 h.

Comparative results of combination studies with BSI-201 and combination studies with Compound A in MDA-MB-231 cells are given in Table 7.

Table 7: Percentage inhibition of combination with BSI-201 and Compound A in

MDA-MB-231 cells

The results indicate that Compound A is more promising than BSI-201 in enhancing the cytotoxicity induced by Gemcitabine and Carboplatin in TNBC MDA-MB-231 cell line.

The synergistic effects in TNBC MDA-MB-231 cell line have been evaluated using the CompuSyn software by Chou and Talalay, described in Pharmacological Reviews, 2006, 58, 621-681. Combination index (CI) is used to evaluate if a combination is additive, synergistic or antagonistic. CI<1 is synergistic, CI=1 is additive and CI>1 is antagonistic. The combination index as evaluated for the combination groups of Regimen 2A and Regimen 2B is shown in Table 8. Table 8: CI values for combination groups of Regimen 2A and Regimen 2B

The results of both the regimens used in the triple drug combination experiments indicated that Compound A is synergistic when used in combination with:

1) Gemcitabine followed by Carboplatin followed by Compound A, and

2) Gemcitabine and Carboplatin together followed by Compound A.

Example 17:

Efficacy profile of triple drug combination of Compound A with Gemcitabine and Carboplatin in triple negative breast cancer xenograft model in SCID mice

Objective: The objective of this study was to evaluate the antitumor activity of Compound A in combination with Gemcitabine and Carboplatin in triple negative human breast cancer xenograft model of MDA-MB-231 (breast adenocarcinoma).

Animal Ethics:

Animals were housed and cared for in accordance with the Guidelines in force published by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Tamil Nadu, India. Procedures using laboratory animals were approved by the IAEC (Institutional Animal Ethics Committee) of the Research Centre of Piramal Life Sciences Limited, Mumbai, India. Method: Human breast adenocarcinoma, MDA-MB-231 cells were grown in RPMI 1640 medium containing 10% fetal calf serum in 5 % CO₂ incubator at 37 °C. Cells were pelleted by centrifugation at 1000 rpm for 10 minutes. Cells were resuspended in a prechilled mixture of saline and matrigel (ratio 1 : 1) to get a count of 7 x 10⁶ cells per 0.2 mL solution and maintained on ice. This suspension was injected by subcutaneous (s.c.) route in female SCID mice (5-6 weeks old). Mice were observed every alternate day for palpable tumor mass.

Once the tumor size reached a size of 3-5 mm in diameter, animals were randomized into respective groups of treatment, Gl to G6 including untreated control. Compound A, BSI- 201, Gemcitabine and Carboplatin were administered i.p as per the regimen given in Table 9, with tumor measurement done every 2-3 days apart. Growth inhibition percentage (Gl %) was calculated at the end of the experiment.

Administration: The schedule of administration in the respective groups of treatment, Gl to G6 is shown in Table 9.

Group Assignments and Dose Levels (D: Day, G: Gemcitabine, C: Carboplatin)

Table 9: Injection volume: 10 mL/kg body weight mpk: mg/kg

Group Anticancer Dose (Route) Regimen

agents

/Control

Gl Compound A 35 mpk (i.p.) D1-D5, D8-D12, D15-D19

G2 BSI-201 50 mpk (i.p.) D1, D4, D8, D11, D15, D18

G3 Gemcitabine + 10 mpk + D1, D8, D15

Carboplatin 10 mpk (i.p.)

G4 Gemcitabine + 10 mpk + G+C: D1, D8, D15

Carboplatin + 10 mpk (i.p.) + Compound A: D1-D5, D8-D12, D15- Compound A 35 mpk (i.p.) D19

G5 Gemcitabine + 10 mpk + G+C: D1, D8, D15

Carboplatin + 10 mpk (i.p.) + BSI-201: Dl, D4, D8, Dl l, D15, D18 BSI-201 50 mpk (i.p.)

G6 Distilled water (control) Dosing:

All mice were intraperitoneally dosed as per study design. Tumor measurement:

a) Tumor weight in milligram was calculated using the formula for a prolate ellipsoid:

Tumor weight (mg) = Length (mm) x [Breadth (mm)²] x 0.5 b) Treated to control ratio (T/C %) for group G4 (treatment of tumor with gemcitabine + carboplatin + Compound A) on a given day X was calculated using the formula:

Ax - Ao

T/C % on day X = x 100

Cx - Co

wherein:

Ax is the tumor size of group G4 (treatment with gemcitabine + carboplatin + Compound A) on day X;

Ao is the tumor size of group G4 (treatment with gemcitabine + carboplatin + Compound A) on day 0;

Cx is the tumor size of group G6 (treatment with control) on day X; and

Co is the tumor size of group G6 (treatment with control) on day 0.

The treated to control ratio was similarly calculated for groups Gl, G2, G3 and G5. c) Growth inhibition (Gl) was calculated as

Gl on day X = 100 - T/C % on day X

Results with

Gl % > 50% drug is considered as active;

Gl % > 75% drug is considered as very active; and

Gl % < 50% drug is considered as inactive.

Results: The results are depicted in Figures 16, 17 and 18. Conclusion:

The combination of Compound A with Gemcitabine and Carboplatin, showed significant in vivo antitumor activity in triple negative breast cancers xenograft models of MDA-MB-231.

Compound A in combination with Gemcitabine and Carboplatin showed antitumor activity with GI = 96 % as compared to BSI-201 in combination with Gemcitabine and Carboplatin showing GI = 82 %. In combination Compound A greatly enhanced the efficacy of Gemcitabine and Carboplatin as compared to only Gemcitabine and Carboplatin combination or Compound A alone.

All the treated groups do not show significant weight loss indicating that the drugs and their combinations are well tolerated.

The invention has been described 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.

Claims

We claim:

1. A pharmaceutical combination for use in the treatment of triple negative breast cancer, wherein said pharmaceutical combination comprises two cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts and a CDK inhibitor selected from the compounds of formula I or a pharmaceutically acceptable salt thereof;

Formula I wherein Ar is phenyl, which is unsubstituted or substituted by 1 , 2, or 3 identical or different substituents selected from: halogen selected from chlorine, bromine, fluorine or iodine; nitro, cyano, Ci-C₄-alkyl, trifluoromethyl, hydroxyl, Ci-C₄-alkoxy, carboxy, C1-C₄- alkoxycarbonyl, CONH₂ or NR1R₂; wherein Ri and R₂ are each independently selected from hydrogen or Ci-C₄-alkyl. 2. The pharmaceutical combination for the use according to claim 1, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by 1 ,

2, or 3 identical or different substituents selected from: halogen selected from chlorine, bromine, fluorine or iodine; Ci-C₄-alkyl or trifluoromethyl.

3. The pharmaceutical combination for the use according to claim 1 or 2, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by 1 , 2, or 3 halogens selected from chlorine, bromine, fluorine or iodine.

4. The pharmaceutical combination for the use according to any one of the claims 1 to 3, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by chlorine.

5. The pharmaceutical combination for the use according to claim 4, wherein the CDK inhibitor is (+)-ira«i-2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl- 1-methyl- pyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A).

6. The pharmaceutical combination for the use according to claim 1 or 2, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is disubstituted with a chloro and a trifluoromethyl group. 7. The pharmaceutical combination for the use according to claim 6, wherein the CDK inhibitor is (+)-ira«i-2-(2-Chloro-4-trifluoromethylphenyl)-5,

7-dihydroxy-8-(2- hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (compound B) .

8. The pharmaceutical combination for the use according to any one of the claims 1 to 7, wherein the cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts; and the CDK inhibitor represented by a compound of formula I or a pharmaceutically acceptable salt thereof; are administered sequentially to a subject in need thereof.

9. The pharmaceutical combination for the use according to claim 8, wherein, the cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts are administered prior to the CDK inhibitor represented by a compound of formula I or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical combination for the use according to any one of the claims 1 to 9, wherein said combination exhibits therapeutic synergy.

11. A method of treating of triple negative breast cancer in a subject comprising administering to the subject a therapeutically effective amount of the cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts in combination with a therapeutically effective amount of a CDK inhibitor selected from the compounds of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof.

12. The method according to claim 11, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by 1, 2, or 3 identical or different substituents selected from: halogen selected from chlorine, bromine, fluorine or iodine; Ci-C4-alkyl or trifluoromethyl.

13. The method according to claim 11 or 12, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by 1, 2, or 3 halogens selected from chlorine, bromine, fluorine or iodine.

14. The method according to any one of the claims 11 to 13, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is substituted by chlorine.

15. The method according to claim 14, wherein the CDK inhibitor is (+)-ira«s-2-(2-Chloro- phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A).

16. The method according to claim 11 or 12, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof; wherein the phenyl group is disubstituted with a chloro and a trifluoromethyl group.

17. The method according to claim 16, wherein the CDK inhibitor is (+)-ira«i-2-(2-Chloro-4- trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)- chromen-4-one hydrochloride (compound B).

18. The method to any one of the claims 11 to 17, wherein a therapeutically effective amount of cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts and a therapeutically effective amount of the CDK inhibitor represented by a compound of formula I or a pharmaceutically acceptable salt thereof; are administered sequentially to the subject in need thereof.

19. The method according to claim 18, wherein therapeutically effective amount of cytotoxic antineoplastic agents, gemcitabine and carboplatin or their pharmaceutically acceptable salts is administered prior to a therapeutically effective amount of the CDK inhibitor represented by a compound of formula I or a pharmaceutically acceptable salt thereof.

20. The method according to any one of the claims 11 to 19, wherein the cytotoxic antineoplastic agents, gemcitabine and carboplatin and the CDK inhibitor exhibits therapeutic synergy.

21. Use of a pharmaceutical combination as defined in claim 1 for the manufacture of a medicament for use in the treatment of triple negative breast cancer.

22. The use according to claim 21, wherein the CDK inhibitor comprised in the pharmaceutical combination defined in claim 1 is (+)-ira«s-2-(2-Chloro-phenyl)-5,7- dihydroxy-8-(2-hydroxymethyl- 1 -methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride (Compound A).

23. The use according to claim 22, wherein the CDK inhibitor comprised in the pharmaceutical combination defined in claim 1 is (+)-ira«i-2-(2-Chloro-4- trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-l-methyl-pyrrolidin-3-yl)- chromen-4-one hydrochloride (Compound B).