TW201307330A - 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

Drug combination for treating breast cancer

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

Cancer is a general term used to describe diseases of uncontrolled division of abnormal cells. Cancer cells can invade adjacent tissues and can spread to other parts of the body via the bloodstream and lymphatic system. There are different types of cancer such as bladder cancer, breast cancer, colorectal cancer, rectal cancer, head and neck cancer, endometrial cancer, kidney (kidney 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. There are more cancer treatments available than ever before, including chemotherapy, radiation therapy, surgery, hormone therapy, immunotherapy, and gene therapy. Chemotherapy is the most commonly used cancer treatment.
The most widely used chemotherapeutic agents (antitumor agents) include paclitaxel, docetaxel, doxorubicin, etoposide, carboplatin, cisplatin, and topotecan ) and gemcitabine. These anti-tumor agents have been successfully used in the treatment of different cancers. However, by the appropriate time, some cancer patients have been found to develop resistance associated with monotherapy using such standard anti-tumor agents. Tolerance or resistance to drugs represents a major obstacle to successful treatment. Such resistance is often considered to be intrinsic (i.e., present in the early stages of treatment) or acquired (i.e., occurring during the course of chemotherapy). A study involving the exposure of human non-small cell lung cancer cells (NCI-H460) to increasing concentrations has been reported to be resistant to stress and to etoposide, paclitaxel, vinblastine, and pan-Ai The emergence of a new cell line (NCI-H460/R) with cross-resistance (Epirubicin) (J. Chemother., 2006, 18, 1, 66-73). Gemcitabine is considered to be the most clinically active drug for the treatment of pancreatic cancer. However, because of the pre-existing or acquired chemical resistance of cancer cells, it does not significantly improve the symptoms of patients with pancreatic cancer (Oncogene, 2003). , 22, 21, 3243-51).
Another problem that is observed or prevalent in cancer treatment is the severe toxicity associated with most anti-tumor agents. Despite the incidence of resistance associated with traditional anti-tumor agents such as gemcitabine and paclitaxel, as well as severe toxicity, these agents will continue to be important in cancer treatment because of their ability to reduce tumor mass. In order to improve response rates and prevent toxicity associated with traditional anti-tumor agents, new treatments are being evaluated.
One such method is directed to a regimen involving the combination of different anti-cancer agents. An ideal combination chemotherapy regimen may result in increased efficacy, reduced host toxicity, and minimal or delayed drug resistance. When drugs with different toxicities are combined, the desired dose can be used for each drug to help minimize unacceptable side effects. Some anticancer agents have been found to have synergistic effects when used in combination with other anti-tumor agents as compared to monotherapy.
Cyclophosphamide and 5-fluorouracil act synergistically in ovarian clear cell adenocarcinoma cells (Cancer Lett., 2001, 162, 1, 39-48). Combination chemotherapy may also be advantageous in the treatment of advanced cancers that are difficult to treat with monotherapy, radiation or surgery. For example, a combination of paclitaxel and gemcitabine has been reported for the treatment of metastatic non-small cell lung cancer (Cancer, 2006, 107, 5, 1050-1054). Combination chemotherapy with gemcitabine and carboplatin is relatively safe and effective for the treatment of elderly patients with non-small cell lung cancer (Cancer Res. Treat., 2008, 40, 116-120). The combination of gemcitabine plus carboplatin is active in advanced transitional cell carcinoma (TCC) with acceptable toxicity (BMC Cancer, 2007, 7, 98). Treatment with gemcitabine and carboplatin significantly improved progression-free survival in patients with platinum-sensitive recurrent ovarian cancer (Int. J. Gynecol. Cancer, 2005, 15 ( Suppl. 1), 36–41).
Currently, the combination of one or more standard anti-tumor agents (eg, paclitaxel, cisplatin, etc.) and one molecule of target anti-tumor agents for the treatment of cancer has been attempted to improve drug response rates and to address resistance to anti-tumor agents. Molecular target agents (e.g., imatinib mesylate, flavopiridol, etc.) modulate proteins, e.g., kinases that are more specifically associated with cancer cells. Researchers have long demonstrated that members of the cyclin-dependent kinase (CDK) family play a key role in various cellular processes. Up to now, there are 11 members of the CDK family. Among them, CDK1, CDK2, CDK3, CDK4, and CDK6 are known to play important roles in the cell cycle (Adv. Cancer Res., 1995, 66, 181-212). CDK is activated by the formation of non-covalent complexes with cyclins (eg, A-form, B-form, C-form, D-form (D1, D2, and D3) and E-type cyclins). Each isozyme of this family is responsible for specific aspects of the cell cycle (cell delivery messages, transcription, etc.), and certain CDK isoforms are specific to a particular type of tissue. Abnormal manifestations and overexpression of these kinases have been demonstrated in many disease symptoms. Some compounds with 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 trials. Flavopiridol has been found to synergistically enhance the cytotoxic response to traditional cytotoxic anti-tumor agents in different cancer cell lines. For example, the combination of paclitaxel and flavopiridol therapy for lung cancer cells has been reported in Radiother. Oncol., 2004, 71, 2, 213-221, and for gastric cancer therapy has been reported in Mol. Cancer Ther., 2003 , 2, 6, 549-555. PCT Publication No. WO2008139271 discloses CDK inhibitors for the treatment of non-small cell lung cancer and pancreatic cancer ((+)- trans - 2-(2-chlorophenyl)-5,7-dihydroxy-8-(2-hydroxyl) A combination of methyl-1-methyl-pyrrolidin-3-yl)-keto-4-one hydrochloride and a cytotoxic tumor agent such as dexamethasone, paclitaxel, paclitaxel, and gemcitabine.
Although there are many treatment options available for cancer treatment, this disease is still one of the most deadly diseases. Although not all types of cancer are fatal, breast cancer is still a deadly type of cancer. In fact, breast cancer is the most common cancer among women and the fifth most common cause of cancer death. Different forms of breast cancer can have significantly different biological characteristics as well as clinical behavior. Therefore, the patient's breast cancer classification has become a key factor in determining treatment options. Breast cancer patients are divided into three main categories:
(i) Patients with hormone receptor-positive tumors treated with some estrus hormone receptor (ER)-target therapy + chemotherapy;
(ii) a patient with a HER2-positive (HER2+) tumor, in addition, receiving a therapy directed to trastuzumab, or in some cases, HER2 under lapatinib;
(iii) Breast cancer patients with hormone receptors ([ER and progesterone receptor (PR)]-negative and HER2), for which chemotherapy is the only available systemic therapy model.
At present, He Cancer has developed into a target therapy for breast cancer patients. Studies have shown that breast cancer performance profiles exhibit systematic changes and classify breast cancer into five major groups, two of which are ER+ (tubular A and B) and three ER-groups (normal breast type, ERBB2). (Also known as HER2) and "base-like"). Despite responses to traditional preadjuvant and adjuvant chemotherapy regimens, it has been shown that basal-type cohorts are enriched for tumors with hormone receptors and HER2 deficiency, and have more aggressive clinical behavior. , special transfer patterns and poor prognosis. Based on the above, the focus on triple-negative cancer stems from (i) the lack of tailor-made therapy for this group of breast cancer patients and (ii) the overlap with the profile of basal-based cancer is clear (Histopathology, 2008, 52). , 108–118).
Triple negative breast cancer (TNBC), a tumor that is negative for estrogen receptor (ER) and negative for the progesterone receptor (PR) and does not overexpress human epidermal growth factor receptor 2 (HER2), accounts for approximately 15% of breast cancer. In 2008, approximately 170,000 cases were reported worldwide. Triple-negative cancer is significantly more aggressive (metastatic) than tumors associated with other molecular subtypes. TNBC does not exhibit estrogen (ER), progesterone (PR), and HER2 receptors, and as such, they are resistant to currently available target treatments, including hormones and HER2-based therapies. Patients with basal or triple-negative breast cancer had significantly shorter survival after the first metastatic event compared with those with non-primary/non-triple negative. The vast majority of tumors caused by BRCA1 germ cell line mutation carriers have morphological features similar to those described in basal-based cancers and they exhibit triple negative and basal phenotypes.
Triple negative breast cancer constitutes one of the most challenging groups of breast cancer. The only systemic therapy currently available to patients with such cancers is chemotherapy. However, the survival rate of patients with such tumors is still poor and their management may require more aggressive intervention. The development of targeted therapies for triple-negative cancers is quite important. Recent experiments have shown that the multiple (ADP-ribose) polymerase (PARP) inhibitor BSI-201 (currently known as a compound developed by Sanofi-Aventis, Iniparib) is highly potent in TNBC (Maturitas, 2009, 63, 269-274). TNBC is also characterized by elevated PARP. These features suggest that PARP inhibition may make chemotherapy-induced DNA damage possible in TNBC (Community Oncology, 2010, 7, 5, 2, 7-10; Clinical Advances in Hematology and Oncology, 7, 7, 441-443 ).
Although triple-negative breast cancer has been reported to respond to chemotherapy, survival of patients with such tumors is still poor and their management may require more aggressive alternative interventions. Therefore, the development of targeted therapy for biologically informed systemic therapies and triple-negative breast cancer is of the utmost importance and can be confirmed by understanding the complexity of this heterogeneous group of tumors and using combination therapies. Achieved (Histopathology, 2008, 52, 108–118).
In view of the above discussion and consideration of the limited treatment options for treating triple-negative breast cancer, there is still a need for additional treatment options and methods for treating TNBC.

In one aspect, the invention relates to a pharmaceutical composition for use in the treatment of triple-negative breast cancer, the composition comprising two cytotoxic anti-tumor agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof; A cyclin-dependent kinase (CDK) inhibitor of a compound of I (as described herein) or a pharmaceutically acceptable salt thereof.
In another aspect, the pharmaceutical composition of the invention exhibits a synergistic effect in the treatment of triple-negative breast cancer.
In another aspect, the invention relates to a pharmaceutical composition for use in the treatment of triple-negative breast cancer comprising two cytotoxic antineoplastic agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof; and a compound selected from formula I a cyclin-dependent kinase (CDK) inhibitor (as described herein) or a pharmaceutically acceptable salt thereof; wherein the two cytotoxic antitumor agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof The class and the CDK inhibitor are administered continuously.
In another further aspect, the invention relates to a method of treating triple-negative breast cancer in a subject comprising a therapeutically effective amount of two cytotoxic antineoplastic agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof; And a therapeutically effective amount of a cyclin-dependent kinase (CDK) inhibitor selected from a compound of Formula I (as described herein) or a pharmaceutically acceptable salt thereof, for administration to a subject.
In yet a further aspect, the invention relates to the use of a pharmaceutical composition as a medicament for the treatment of triple-negative breast cancer.
Other aspects of the invention, as well as further scope of applicability, will become apparent from the detailed description.

The pharmaceutical composition of the present invention has now been found to comprise two cytotoxic antineoplastic agents, gemcitabine and carboplatin, or a pharmaceutically acceptable salt thereof, and a compound selected from formula I (as described herein) or a pharmaceutical thereof A CDK inhibitor of an acceptable salt; exhibits a synergistic effect when used in the treatment of triple-negative breast cancer.
In particular, the present invention provides a method of treating or treating triple-negative breast cancer in a subject comprising a therapeutically effective amount of two cytotoxic antitumor agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof; and a treatment An effective amount of a cyclin-dependent kinase (CDK) inhibitor selected from a compound of Formula I (as described herein) or a pharmaceutically acceptable salt thereof is administered to the subject.
The CDK inhibitor included in the pharmaceutical composition of the invention is selected from the compounds 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 No. 7,272,193) and PCT Publication No. WO2007148158, which is incorporated herein by reference. The compound of formula I is a CDK inhibitor that inhibits proliferation of different cancer cells. The compounds of formula I as used in the present invention are effective against different solid and hematological malignancies. The inventors of the present invention have observed that a combined CDK inhibitor (a compound of formula I with a cytotoxic antitumor agent (i.e., gemcitabine and carboplatin) causes an increase in apoptosis or stylized cell death.
The CDK inhibitor used in the present invention is selected from the compounds represented by the following formula I,
Formula I

Wherein Ar is a phenyl group which is unsubstituted or substituted by 1, 2 or 3 identical or different substituents selected from the group consisting of halogens selected from chlorine, bromine, fluorine or iodine, Nitro, cyano, , trifluoromethyl, hydroxyl, ,carboxyl, , or ;
Each of them versus Is independently selected from hydrogen or .
Compounds of formula I, including its image-isomerized pure form, can be prepared according to the methods disclosed in PCT Publication No. WO2004004632 and PCT Publication No. WO2007148158, which is incorporated herein by reference.
The general procedure for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises the following steps:
(a) The resolved mirror image isomerized pure (-)-trans mirror image isomer of the intermediate compound of formula VIA in the presence of a Lewis acid catalyst

Treated with acetic anhydride to obtain an acetylated compound of the decomposition of formula VIIA,

(b) reacting the decomposed ethoxylated compound of formula VIIA with an acid of the formula ArCOOH or an acid chloride of the formula ArCOCl or an anhydride of the formula (ArCO) ₂ O or an ester of the formula ArCOOCH ₃ in the presence of a base and a solvent. Wherein Ar is a compound of formula (I) as defined herein above to obtain a decomposing compound of formula VIIIA;

(c) treating the decomposing compound of the formula VIIIA with a base in a suitable solvent to obtain the corresponding decomposed β-diketone compound of the formula IXA;

Wherein Ar is as defined above;
(d) treating the decomposed β-diketone compound of formula IXA with an acid such as hydrochloric acid to obtain the corresponding cyclized compound of formula XA,

(e) by being between At a temperature, the compound of formula XA is heated with a dealkylating agent, and the compound of formula XA is subjected to dealkylation to obtain the (+)- trans mirror isomer of the compound of formula (I), and Alternatively, the compound of interest is converted to its pharmaceutically acceptable salt.
The Lewis acid catalyst utilized in the above step (a) may be selected from the group consisting of: , , zinc chloride, aluminum chloride and titanium chloride.
The base utilized in the procedure step (b) may be selected from the group consisting of triethylamine, pyridine, and DCC-DMAP compositions (combination of N, N'-dicyclohexylcarbenium imine and 4-dimethylaminopyridine) .

It will be apparent to those skilled in the art that the compound of formula VIIIA is recombined into a corresponding beta-diketone compound of formula IXA known as Baker-Venkataraman rearrangement (J. Chem.). Soc., 1933, 1381 and Curr. Sci., 1933, 4, 214) will be apparent to those skilled in the art.
The base used in the procedure step (c) may be selected from the group consisting of lithium hexamethyldiamine, sodium hexamethyldiamine, potassium hexamethyldiamine, sodium hydride and potassium hydride. A preferred base is lithium hexamethyldiamine.
The dealkylating agent used in the dealkylation of the compound of formula IXA in process step (e) may be selected from the group consisting of: pyridine hydrochloride, boron tribromide, boron trifluoride etherate, and aluminum trichloride. . A preferred dealkylating agent is pyridine hydrochloride.
The preparation of the starting compound of formula VIA involves reacting 1-methyl-4-piperidone with a 1,3,5-trimethoxybenzene solution in glacial acetic acid to form 1-methyl-4-(2, 4,6-Trimethoxyphenyl)-1,2,3,6-tetrahydropyridine, which is reacted with boron trifluoride diethyl ether, sodium borohydride and tetrahydrofuran to form 1-methyl-4-(2, 4,6-Trimethoxyphenyl)piperidin-3-ol. Conversion of 1-methyl-4-(2,4,6-trimethoxyphenyl)piperidin-3-ol to a compound of formula VIA involves an oxygen nucleophile such as triethylamine, pyridine, potassium carbonate or sodium carbonate In the presence of a compound such as p-toluenesulfonyl chloride, methanesulfonate chloride, trifluoromethanesulfonic anhydride or phosphorus pentachloride, will appear in the compound 1-methyl-4-( The hydroxyl group on the piperidine ring of 2,4,6-trimethoxyphenyl)piperidin-3-ol is converted to a leaving group (eg toluenesulfonyl, methylsulfonyl, trifluoromethanesulfonic acid or The halide is then subjected to a ring contraction in an alcohol solvent such as isopropanol, ethanol or propanol in the presence of an oxygen nucleophile such as sodium acetate or potassium acetate.
In a particular 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 the group consisting of: chlorine, bromine , halogen or iodine halogen, And trifluoromethyl.
In another specific embodiment, the CDK inhibitor is a compound of formula I wherein the phenyl group is substituted by 1, 2 or 3 halogens selected from the group consisting of chlorine, bromine, fluorine or iodine.
In another specific embodiment, the CDK inhibitor is a compound of formula I wherein the phenyl group is substituted with chlorine.
In a further embodiment, the CDK inhibitor represented by the compound of Formula I is (+)- trans - 2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxyl) Oryl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one or a pharmaceutically acceptable salt thereof.
In a still further embodiment, the CDK inhibitor represented by the compound of Formula I is (+)- trans - 2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxyl Methyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (designated herein as Compound A).
In another specific embodiment, the CDK inhibitor is a compound of formula I wherein the phenyl group is disubstituted by a monochloro and a trifluoromethyl group.
In a further embodiment, the CDK inhibitor represented by the compound of Formula I is (+)- trans - 2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8- ( 2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one, or a pharmaceutically acceptable salt thereof.
In a still further embodiment, the CDK inhibitor represented by the compound of Formula I is (+)- trans - 2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8- (2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (designated herein as Compound B).
In a specific embodiment, the CDK inhibitor represented by the compound of Formula I is an antiangiogenic agent.
In a specific embodiment, the CDK inhibitor represented by the compound of Formula I is a HIF-1α inhibitor. In a specific embodiment, the CDK inhibitor represented by the compound of Formula I is a VEG-F inhibitor. In a specific embodiment, the CDK inhibitor represented by the compound of Formula I is a PARP enzyme inhibitor.
The manufacture of a compound of formula I which may be in the form of a pharmaceutically acceptable salt, and the manufacture of a pharmaceutical composition comprising an oral and/or parenteral composition comprising the above compounds is disclosed in PCT Publication No. WO2004004632 (corresponding to U.S. Patent 7,272,193). And PCT Publication No. WO2007148158. These PCT publications disclose that CDK inhibitors represented by Formula I inhibit proliferation of different cancer cells. As indicated herein above, the CDK inhibitors of Formula I can be used in their salt form. Preferred salts of the compounds of formula I include the hydrochloride, methanesulfonate and trifluoroacetate salts.
The compound of formula I contains at least two palmitic centers and is therefore present in the form of two different optical isomers (i.e., (+) or (-) mirror image isomers). All such mirror image isomers and mixtures thereof, including racemic mixtures, are included within the scope of the invention. The mirror image isomers of the compounds of formula I can be obtained by the methods disclosed in PCT Publication Nos. WO2004004632, WO2008007169, and WO2007148158, or the mirror image isomers of the compounds of Formula I can also be obtained by methods well known in the art, such as palm chromatography and Enzymatic resolution. The phrase "enantiomerically pure" describes compounds which are present in greater than 95% enantiomeric excess (ee). In another embodiment, the mirror image isomer excess value is greater than 97%. In yet another embodiment, the image isomer excess value is greater than 99%. The phrase "mirror isomer excess" describes the difference between the amount of one mirror isomer present in the product mixture and the amount of another mirror image isomer.
Alternatively, the mirror image isomer of the compound of formula I can be synthesized by using an optically active starting material. Thus, a compound of formula I is defined to encompass all possible stereoisomers and mixtures thereof. The definition of a compound of formula I includes racemic forms as well as isolated optical isomers with specific activities.
The two cytotoxic antineoplastic agents used in the pharmaceutical compositions of the invention are selected from the group consisting of commercially available gemcitabine and carboplatin.
Gemcitabine is a common name designated as 2'-deoxy-2',2'-difluorocytosine. It is commercially available as a monohydrochloride as well as a beta-isomer. The gemcitabine is disclosed in U.S. Patent Nos. 4,808,614 and 5,464,826, the disclosures of each of each of each of each of each of each The commercial formulation of gemcitabine hydrochloride as a single agent has been shown to be the first line of treatment for patients with locally advanced pancreatic metastatic or metastatic adenocarcinoma or lung cell carcinoma (NSCLC) and is commonly used in previous 5- Patients treated with fluorouracil.
Carboplatin is a common name for platinum designated as cis-diamine (1,1-cyclobutanedicarboxylic acid). Carboplatin was discovered and developed at the Cancer Institute in London. Carboplatin is disclosed in U.S. Patent No. 4,657,927, the disclosure of which is incorporated herein by reference in its entirety in its entirety in its entirety in the in the in the in the Carboplatin kills cancer cells by binding to DNA and interfering with the repair mechanisms of the cells, which ultimately leads to cell death. It is classified as an alkylating agent. It is considered to be the "second generation" platinum-based agent. Carboplatin is chemically different from cisplatin because it is a larger molecule with a dicarboxylic acid ligand. This slows down the metabolic breakdown of the agent (which stays longer in the body) and reduces the rate of formation of toxic by-products. Carboplatin is used to treat ovarian cancer. Carboplatin is also used in other types of cancer, including the lungs, head and neck, endometrium, esophagus, bladder, breast, and cervical cancer; central nervous system or germ cell tumors; osteogenic sarcoma; Preparation of stem cells or bone marrow transplants.
The general terms used above and below preferably have the following meanings within the context of the disclosure, unless otherwise stated:
The term "antineoplastic agent" is synonymous with "chemotherapeutic agent" or "anticancer agent" and means a therapeutic agent that acts by inhibiting or preventing the growth of a tumor. The term "anti-tumor agent" or "anti-cancer agent" generally means a compound that prevents cancer cells from multiplying (ie, an anti-proliferative agent). In general, anti-tumor agents fall into two categories, anti-proliferative cytotoxicity and anti-proliferative cell growth inhibitors. Anti-proliferative cytotoxic agents prevent cancer cell proliferation by: (1) interfering with the ability of cells to replicate DNA and (2) inducing cell death and/or apoptosis in cancer cells. Anti-proliferative cell growth inhibitors function by modulating, interfering with, or inhibiting the process of cell signaling that regulates cell proliferation. The antitumor agent contained in the pharmaceutical composition of the present invention in the present invention is a cytotoxic agent and thus means a cytotoxic antitumor agent.
As used herein, the term "composition" or "pharmaceutical composition" means two cytotoxic antitumor agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof, and a CDK inhibitor (a compound of formula I). The combination is administered; the therapeutic agent can be administered separately independently at the same time or in a time interval in which the combination partner specifically exhibits a synergistic effect.
As used herein, the term "synergistic" means that the effects achieved by the methods and compositions of the present invention are greater than due to the use of an antitumor agent, or a pharmaceutically acceptable salt thereof, and a CDK inhibitor, respectively. The sum of the effects of a compound of formula I or a pharmaceutically acceptable salt thereof. Advantageously, such synergistic effects provide greater efficacy at the same dose and/or avoid or delay the formation of multiple drug resistance.
Reference to "therapeutically effective amount" of treatment of triple-negative breast cancer means an amount that is capable of causing one or more of the following effects in a subject receiving the composition of the invention: (i) inhibition to a certain degree of tumor growth, including Slowing down and complete growth arrest; (ii) reduction in the number of cancer cells; (iii) reduction in tumor size; (iv) inhibition of tumor cell infiltration to surrounding organs (ie, reduction, slowing or complete cessation); (v) metastasis Inhibition (ie, reduction, slowing or complete cessation); (vi) enhancement of the anti-tumor immune response, which may, but not necessarily, cause regression or rejection of the tumor; and/or (vii) to some extent One or more of the symptoms associated with triple-negative breast cancer.
As used herein, the terms "manage", "managing" and "management" mean a subject or patient derived from a pharmaceutical composition of the invention, when administered to said A patient or subject so as to prevent the beneficial effects of progression or deterioration of TNBC.
As used herein, the term "triple-negative breast cancer" or "TNBC" encompasses malignant tumors of different histopathological phenotypes. For example, some TNBC classifications are referred to as "basal-like"("BL"), in which tumor cells exhibit genes frequently found in normal basal/myoepithelial cells of the breast, such as high molecular weight basal keratin. Cytokeratin (CK, CK5/6, CK14, CK17), vimentin, p-cadherin, ccB crystallin, actin fascin, and Caveolins 1 and 2. However, some other TNBCs have different histopathological phenotypes, examples of which include high grade invasive ductal carcinoma, metaplastic carcinomas, and medullary carcinoma (without specific types of breasts). Medullary carcinomas) and salivary gland-like tumors. The TNBC provided for use in the pharmaceutical compositions of the present invention may be non-reactive or refractory TNBC.
As used herein, the term "non-responsive/refractory" is used to describe a subject with triple negative breast cancer (TNBC) or a cancer therapy that has been treated with currently available TNBC ( For example, chemotherapy, radiation therapy, surgery, hormonal therapy, and/or biological therapy/immunotherapy, where the therapy is not clinically sufficient to treat the patient, so that these patients require additional effective therapies, for example, to maintain therapy Not susceptible. The term may also describe a subject or patient who is responsive to therapy, also suffering from side effects, recurrence, development of resistance, and the like. In various embodiments, "non-reactive/refractory" means that at least some significant portions of cancer cells are not killed or their cell division is arrested. Whether the cancer cells are "non-reactive/refractory" can be determined in vivo or in vitro by any method known in the art for analyzing the therapeutic effect on cancer cells, and in this context, the field of "refractory" is accepted. significance. When the number of cancer cells is not significantly reduced or increased, the cancer is "non-reactive/refractory".
As used herein, the term "treatment cycle" means the administration of a cytotoxic antineoplastic agent or a pharmaceutically acceptable salt thereof, and a CDK inhibitor (ie, a compound of formula I) or a pharmaceutically acceptable salt thereof. The time period of the cycle of the drug.
The term "apoptosis" means a form of cell death in which a series of molecular steps in a cell causes its death. This is the normal way for the body to eliminate unwanted or abnormal cells. The process of apoptosis in cancer cells may be blocked. Also known as programmed cell death (National Cancer Institute Cancer Dictionary).
As used herein, the term "increased apoptosis" is defined as an increase in the rate of programmed cell death, ie, when exposed to a composition of an antitumor agent alone or a CDK inhibitor alone (contact). More cells are induced to the death program.
As used herein, the term "subject" means an animal, preferably a mammal, and most preferably a human, which is the subject of treatment, observation or experimentation.
In a specific embodiment, the invention relates to a pharmaceutical composition for the treatment of triple-negative breast cancer, wherein the composition comprises two cytotoxic anti-tumor agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof, and At least one cyclin dependent kinase (CDK) inhibitor, or a pharmaceutically acceptable salt thereof, selected from a compound of Formula I (as described herein).
In a specific embodiment, a pharmaceutical composition comprising a CDK inhibitor (i.e., a compound of Formula I and a cytotoxic antineoplastic agent as described herein) comprises those that permit separate administration, which may be administered continuously or at intervals of time. Get the maximum effect of the combination. Thus, for effective cancer treatment, the drug conjugates can be administered separately or periodically, for a period of time.
In a particular embodiment of the invention, the cytotoxic antineoplastic agent, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof, is a CDK inhibitor (ie, a compound of formula I or a pharmaceutically acceptable salt thereof) Dosing prior to administration.
In a specific embodiment, the CDK inhibitor included in the drug conjugate of the invention is selected from Compound A or Compound B.
In a specific embodiment, the therapeutic agent (ie, the cytotoxic anti-tumor agent and the CDK inhibitor contained in the conjugate) may have to be administered by different routes due to their different physical and chemical properties. For example, a CDK inhibitor of a compound of Formula I can be administered orally or parenterally to produce and maintain a good blood concentration, while an anti-neoplastic agent can be administered parenterally via an intravenous, subcutaneous or intramuscular route.
For oral use, the CDK inhibitor of the compound of formula I can be administered, for example, in the form of a lozenge or capsule, a powder, a dispersible granule or a pharmaceutical pack or in the form of an aqueous solution or suspension. In the case of tablets for oral use, conventional carriers include lactose, corn starch, magnesium carbonate, talc and sugar, and a lubricant such as magnesium stearate is often added. For oral administration in the form of a capsule, conventional carriers include lactose, corn starch, magnesium carbonate, talc, and sugar.
As an intramuscular, intraperitoneal, subcutaneous, and intravenous injection, a sterile solution of the active ingredient (antitumor agent or CDK inhibitor) is usually employed, and the pH of the solution should be appropriately adjusted and buffered.
In a specific embodiment, the sterile solution of the active ingredient used is prepared in physiological saline or distilled water.
In a specific embodiment, the invention relates to a method of treating triple-negative breast cancer in a subject comprising a therapeutically effective amount of two cytotoxic anti-tumor agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof A therapeutically effective amount of a CDK inhibitor selected from a compound of Formula I or a pharmaceutically acceptable salt thereof, is administered for administration to the subject.
Thus, in the methods of the invention, a therapeutically effective amount of two cytotoxic antineoplastic agents effective for treating cancer is treated with a therapeutically effective amount of a compound selected from Formula I, or a pharmaceutically acceptable salt thereof, The CDK inhibitor is administered to the subject in combination, while the subject treats triple-negative breast cancer, wherein the combined use of the therapeutic agent exhibits a synergistic effect.
In a specific embodiment, the method of treating triple negative breast cancer in a subject comprises administering a therapeutically effective amount of a cytotoxic antineoplastic agent, gemcitabine and carboplatin, and a therapeutically effective amount of a CDK inhibitor (represented by a compound of formula I) The conjugate is administered to the subject, wherein the cytotoxic antineoplastic agent, gemcitabine and carboplatin, and the CDK inhibitor are administered continuously.
In another embodiment, the method of treating triple negative breast cancer in a subject comprises administering a therapeutically effective amount of cells prior to administration of a CDK inhibitor selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt thereof. A toxic anti-tumor agent, gemcitabine and carboplatin, was administered to the subject.
In a specific embodiment, the method of treating triple-negative breast cancer in a subject comprises administering gemcitabine and carboplatin to the subject continuously, followed by a CDK inhibitor selected from a compound of formula I or a pharmaceutically acceptable salt thereof. Administration.
In a specific embodiment, the method of treating triple-negative breast cancer in a subject comprises administering gemcitabine concurrently with carboplatin, followed by administration of a CDK inhibitor selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt thereof Apply to the subject.
In a specific embodiment, the CDK inhibitor used in the method of treating triple-negative breast cancer is selected from Compound A or Compound B.
In a specific embodiment, the invention relates to the use of a pharmaceutical conjugate for the manufacture of a medicament for the treatment of triple-negative breast cancer, wherein the pharmaceutical conjugate comprises two cytotoxic anti-tumor agents, gemcitabine and carboplatin, and A CDK inhibitor of a compound of formula I or a pharmaceutically acceptable salt thereof.
The actual dose of the anti-cancer agent contained in the conjugate can vary depending on the needs of the patient and the severity of the condition being treated. In general, the treatment begins with a smaller dose that is less than the optimal dose of the compound. Thereafter, the dose of each anticancer agent is increased in small amounts until the best effect in this case is achieved. However, the dose of each anti-cancer agent in the drug conjugate will typically be less than the amount that would produce a therapeutic effect if administered alone. For convenience, the total daily dose may be divided and partially administered during the day if needed. In a specific embodiment, the two cytotoxic antineoplastic agents, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof, and a CDK inhibitor system selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt thereof Continuous administration in an injectable form such that each cytotoxic anti-tumor agent is to a range of synergistically effective doses, and CDK inhibitors are to Range of doses, in particular To approximately Dosage of the range of doses.
In a specific embodiment, a pharmaceutical conjugate system for 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 :

In a specific embodiment, the drug conjugate is administered to a subject in two to six treatment cycles, before or after surgery, or partially before surgery, and partially after surgery.
The combinations provided by the present invention have been evaluated in certain test systems as well as in several different in vitro dosing schedules. Experimental details are provided below. The information presented herein clearly indicates two cytotoxic anti-tumor agents, gemcitabine and carboplatin exhibit a synergistic effect when combined with a CDK inhibitor selected from a compound of formula I. It clearly states that when an anticancer agent is used in a combination of triple-negative breast cancer treatment, the anti-resistance is compared to when the cell is treated only with a CDK inhibitor (ie, a compound of formula I alone or a cytotoxic anti-tumor agent alone) Cancer agents increase apoptosis or cytotoxicity in proliferating cells.
A representative compound, Compound A used in the pharmacological test means (+)- trans - 2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl) Is a compound of the disclosed PCR Publication No. WO2004004632, which is incorporated herein by reference.
The inventors have also established xenograft modes to extend in vitro observations to in vivo systems. The inventors tested the in vivo effects of the conjugates of the invention in male mice of SCID mice (Severely Combined Immune Deficient) using a triple negative breast cancer xenograft mode. It has been observed that when administered in combination with a cytotoxic drug conjugate, the CDK inhibitor synergistically enhances the effect of the cytotoxic drug conjugate of gemcitabine and carboplatin.
The synergy of the conjugates of the invention comprising two cytotoxic antineoplastic agents (gemcitabine and carboplatin) and a CDK inhibitor is now explained in more detail with reference to specific embodiments thereof. It is to be noted that these are provided as examples only and are not intended to limit the invention.

The following abbreviations or terms are used in this article:

Cell line (Source: ATCC, USA):

Cell line (Source: NCI, USA):
antibody ( source : Cell Signaling Technology, USA ):

The invention is further described by the following non-limiting examples, which are not intended to be construed as limiting the scope of the invention.

example:

Example 1:
(+) - trans - 2- (2-chlorophenyl) -5,7-dihydroxy-8- (2-hydroxymethyl-1-methyl - pyrrolidin-3-yl) - g Xi -4- Preparation of ketohydrochloride (Compound A) sodium hydride (50%, 0.54 g, 11.25 mmol) Under nitrogen atmosphere and added portionwise to stirred in dry DMF (15 mL) in the (-) - trans - 1- [2-hydroxy-3- (2-hydroxymethyl-1-methyl-3-pyrrolidin A solution of 4,6-dimethoxyphenyl)-ethanone (0.7 g., 2.2 mmol). After 10 minutes, methyl 2-chlorobenzoate (1.15 g., 6.75 mmol) was added. The reaction mixture is Stir under 2 hours. Below Carefully add methanol. The reaction mixture was poured on EtOAc (EtOAc) (EtOAc) Use saturation Alkaline the water layer and use (3 x 200 mL) extraction. Dry and concentrate the organic layer (anhydrous ). Concentrated HCl (25 mL) was added to the residue and stirred at room temperature for 2 hr. The reaction mixture was poured on crushed ice (300 g) and saturated The aqueous solution is alkalized. use (3 x 200 mL) extract the mixture. The organic extract is rinsed with water and dried (anhydrous And concentrated to obtain the compound (+)- trans - 2-(2-chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7- Dimethoxy-keken-4-one.

The molten pyridine hydrochloride (4.1 g, 35.6 mmol) was added to (+)-anti- -2-(2-Chloro-phenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-c-c--4-one (0.4 g, 0.9 mmol), andHeat for 1.5 hours. Cool the reaction mixture to, diluted with MeOH (10 mL) and usedAlkalinize 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 give compound (+)-anti- -2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-g-indolin-4-one. [Yield: 0.25 g (70%)]
Will (+)-anti- -2-(2-Chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glycan-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 give a clear solution. The solution is cooled and the solid is filtered off to obtain the compound (+)-anti- -2-(2-Chlorophenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride or compound A..

Example 2:
(+)-anti- -2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methylpyrrolidin-3-yl)-c- Preparation of 4-ketohydrochloride (Compound B)
Willanti- -1-[2-Hydroxy-3-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-4,6-dimethoxyphenyl)-ethanone (1.16 g, 3.2 mmol), 2 a mixture of -chloro-4-trifluoromethylbenzoic acid (0.88 g, 4 mmol), DCC (1.35 g, 6.5 mmol) and DMAP (0.4 g, 3.27 mmol) dissolved in dichloromethane (50 mL) Stir at room temperature for 12 hours. Cool the reaction mixture toThe precipitated dicyclohexylurea was filtered, and the organic layer was concentrated and purified by column chromatography using 1% methanol in chloroform and 0.01% aqueous ammonia as a solvent to obtain compound (+)-anti- -2-(2-Ethyloxymethyl-1-methyl-pyrrolidin-3-yl)-6-ethylindolyl-3,5-dimethoxybenzene 2-chloro-4-trifluoromethylbenzoate Base ester [yield: 1.44 g (78.8 %)].
Hexamethyldioxane (1.08 mL, 5.1 mmol) was added dropwise to a solution of n-BuLi (15% in hexanes) in THF (10 mL) maintained at 0 ° C under nitrogen atmosphere. 2.2 mL, 5 mmol) and stir for 15 minutes. (+)- in THF (10 mL)anti- -2-(2-Ethyloxymethyl-1-methyl-pyrrolidin-3-yl)-6-ethylindolyl-3,5-dimethoxybenzene 2-chloro-4-trifluoromethylbenzoate The base ester solution (1.44 g, 2.5 mmol) was added dropwise to maintain the temperature. After the addition, the reaction was allowed to warm to room temperature and stirred for 2.5 hours. The reaction mixture was acidified with dilute HCl and basified to pH 8 to 9 with 10% sodium bicarbonate. The aqueous layer was extracted with chloroform (3 x 25 mL). Rinse the organic layer with water (25 mL), brine (25 mL), and drydry. The organic layer was concentrated under reduced pressure and dried in vacuo to afford crystals of 3-(3-[3-(2-chloro-4-trifluoromethyl-phenyl)-3-oxo- Propionyl]-2-hydroxy-4,6-dimethoxy-phenyl}-1-methyl-pyrrolidin-2-ylmethyl acetate (1.3 g, 90.2%). This ester was dissolved in concentrated HCl (10 mL) and stirred for 3 hours to cause cyclization. At the end of 3 hours, the reaction mixture was solidBasic to pH 8 to 9. The aqueous layer was extracted with chloroform (25×3 mL) and rinsed with water (25 mL) and brine (25 mL). The organic layer is anhydrousDry, concentrate under reduced pressure and dry under vacuum. The residue was purified by column chromatography, 3% methanol in chloroform and 0.1% aqueous ammonia as a solvent to give a compound as a yellow solid (+)-anti- -2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-5,7-dimethoxy-g--4- ketone. [Yield: 0.56 g (48.2 %)]
Will (+)-anti- -2-(2-Chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-5,7-dimethoxy-g--4- a mixture of ketone (0.25 g, 0.5 mmol), pyridine hydrochloride (0.25 g, 2.16 mmol) and a catalytic amount of quinolineHeat for a period of 2.5 hours. The reaction mixture was diluted with methanol (25 mL) and taken a solid Na₂CO₃Alkalinize to pH 10. The reaction mixture was filtered and washed with methanol. The organic layer was concentrated and purified by column chromatography, using 0.1% aqueous ammonia in chloroform and 4.5% methanol as eluent to give a compound as a yellow solid (+)-anti- -2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methylpyrrolidin-3-yl)-c-c--4-one . [Yield: 0.15 g (63.7 %)]
Will (+)-anti- -2-(2-Chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxy-methyl-1-methylpyrrolidin-3-yl)-ketone-4-one (0.1 g, 0.2 mmol) was suspended in MeOH (2 mL).anti- -2-(2-Chloro-4-trifluoromethyl-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-c- 4-keto hydrochloride. [Yield: 0.1g (92.8 %)]

Pharmacological test:

example 3 :
Use propidium iodide ( PI Cytotoxicity test
The Propidium iodide Fluorescence Test (PI) was carried out according to the procedure outlined in Anticancer Drugs, 1995, 6, 522-532.
This assay was developed to characterize the in vitro growth characteristics of human tumor cell lines as well as the cytotoxic activity of test compounds. Propidium iodide (PI) is used as a dye that penetrates only the damaged cell membrane. The embedded complex that causes amplification of the fluorescence is formed by PI and double stranded DNA. In the cellAfter 24 hours of freezing, PI can pass to the overall DNA resulting in a total cell population count. Background readings were obtained from cell-free wells containing medium and propidium iodide.
Human breast cancer cell lines (ie MCF-7, T47-D, ZR-75-1, MDA-MB-468, MDA-MB-231, MDA-MB-435-S, MDA-MB-361, HBL-100) , BT-549) implanted into a 96-well plate at a density of 1500-3000 cells/well in 180 μL of DMEM (Dulbecco's Modified Eagle's Medium, Gibco, USA) or RPMI 1460 with 10% FCS, and cultured for approximately 16 hours. Attach the cells. The cells were then treated with different concentrations of Compound A (0.1 to 3 μM). The above procedure was repeated at three concentrations of Compound A, Gemcitabine, Carboplatin and BSI-201 (Iniparib developed by Sanofi-Aventis) in three TNBC cell lines (MDA-MB-231, MDA-MB-468 and BT-549). That is, the concentration of Compound A and Gemcitabine (Tocris, UK) in the total time of 48 hours ranged from 0.01 to 3 μM, and for Carboplatin (Shandong Boyuan Chemical Co. Ltd., China) and BSI-201 (manufactured internally) The concentration range is 10-300 μM. Cultivate the plate inWetIncubator. The wells of the control group were treated with vehicle (DMSO). At the end of the incubation period, the PI plate was used to analyze the plates. Calculate the percentage of cytotoxicity at different drug concentrations and determine from the plotted plotValue. The results of this study are shown in Tables 1A and 1B.

table 1A :
Antiproliferative activity of compound A, BSI-201, carboplatin and gemcitabine against TNBC

Table 1A shows that Compound A showed significantly higher anti-proliferative potential when compared to the target drug (i.e., BSI-201 and carboplatin).

table 1B :
Anti-proliferative potential of Compound A as measured by PI assay in different breast cancer cell lines (IC in μM)₅₀)


Table 1B shows that Compound A was found to be between 0.3 and 1.0 μM for breast cancer cell lines regardless of genetic markers.The range is effective against proliferation.

example 4 :
Adult test or cell colony formation test

MDA-MB-231, MDA-MB-468 and MCF-7 cell lines were seeded in a six-well plate at a density of 1500 cells/well at RPMI 1460 with 10% FCS. After 24 hours of incubation, the cells were, IC₃₀And IC₅₀Concentration of Compound A (as determined by the procedure of Example 3) for a period of 48 hours and will , IC₃₀And IC₅₀The values are presented in Table 2. The medium was removed at the end of the treatment and cultured for 14 days in fresh medium (without drug). The medium was aspirated after 14 days and the cell population was fixed with a 2:1 ratio of methanol to acetic acid mixture, wetted with water and the fixation procedure repeated. The dish was dried and the cell population was stained with 0.1% crystal violet for 5 minutes. Finally the holes are wetted with water and dried.

Table 2:

The results are depicted in Figure 1, which is via Compound A in MDA-MB-231, MDA-MB-468, and MCF-7 cell lines (planting density: 1500 cells/well)., IC₃₀And IC₅₀The response of the dose shows a visual enhancement.
Compound A was found to inhibit cell community formation potential in a dose-dependent manner.

example 5 :
Effect of Compound A on the formation of multicellular tumor spheres (3D)
This test was carried out according to the method disclosed in Methods in Molecular Medicine, 2007, 140, 141-151.
The multicellular tumor sphere (MCTS) model is one of the best described 3D in in vitro tumor model systems, which characterizes many of the characteristics of tumor tissue and recognizes reproducible experiments, providing an excellent in vitro screening system. MCTS was propagated using the hanging drop method. Briefly, cell monolayers were detached using trypsin-EDTA. The cell count was adjusted and a 20 μL hanging drop containing 1,000 cells/drop was made in a bacterial grade dish. Hang these drops inUnder humid air at 37^oC was cultured for 24 hours. The MCTS thus produced was cultured for 72 hours in the presence or absence of different concentrations of Compound A (0.3 μM to 30 μM).
The results are presented in Figure 2.
When the MCF-7 cell suspension was co-cultured with different concentrations of Compound A (0.3 μM to 30 μM) in MCTS proliferation, spheroid formation was arrested from Compound A at a concentration of 3 μM. The size of the MCTS formed at 1 μM was also small compared to the control group. This observation is important from a clinical point of view because the MCTS system is sufficiently characterized by a pathological environment that mimics a patient's tumor. It mimics the microenvironment that is widespread in tumor tissue due to the oxygen gradient that causes tumor hypoxia in the sphere. The effect of Compound A on the formation of spheres indicates that Compound A is effective in the absence of oxygen.

example 6 :
Time-dependent effects of Compound A on cell cycle progression and apoptosis in MCF-7 (Her low, ER+, PR+, BRCA +/- dual gene loss) and TNBC cell line MDA-MB-231
Time-dependent effects of Compound A on cell cycle progression and apoptosis were evaluated in two breast cancer cell lines. Non-synchronized human breast cancer cell line MCF-7 (Her low, ER+, PR+, BRCA +/- dual gene loss) and MDA-MB-231 with RPMI 1460 with 10% FCS, per bottleThe density of cells is planted inTissue culture flasks. After 24 hours, the cells were treated with 4.5 μM of Compound A for 0, 24, 48 and 72 hours. Both desorbed and attached cells were collected (digested by trypsin) at various time points as described in Table 3. After washing in phosphate buffered saline (PBS), the cells were fixed in ice-cold 70% ethanol and stored at -20 °C until further analysis.
Prior to analysis, cells were washed twice with PBS to remove the fixative and resuspended in PBS containing 50 μg/mL propidium iodide and 50 μg/mL RNaseA. At room temperatureAfter 20 minutes of culture, the cells were analyzed using a flow cytometer. These studies used a Becton Dickinson FACS Calibur flow cytometer (BD, USA). An argon ion laser set at 488 nm was used as the excitation source. Cells with a DNA content between 2n and 4n were designated as in the G1, S and G2/M phases of the cell cycle as defined by the degree of red fluorescence. Cells expressing less than 2n DNA content were designated as sub-G1 (apoptotic population) cells. The number of cells separated at each cell cycle is expressed as a percentage of the total amount of cells present. The results are shown in Table 3 and are shown graphically in Figure 3A (MCF-7 cell line) and Figure 3B (MDA-MB-231 cell line).

Table 3: Percentage of apoptosis

From the results shown in the above table, Compound A was induced to induce apoptosis in MCF-7 (Her low, ER+, PR+, BRCA +/- dual gene loss) and TNBC cell line MDA-MB-231. The highest apoptosis was seen at 48 hours and 72 hours.

example 7:
The effect of Compound A on MCF-7 and MDA-MB-231 cells was analyzed using Western blots:
Western blot analysis was performed according to the procedure disclosed in Molecular Cancer Therapeutics, 2007, 6, 918-925, and some modifications.
MCF-7 and MDA-MB-231 cells were planted in RPMI 1460 with 10% FCSTissue culture flasks were incubated for 24 hours. Cells were treated with 1.5 and 4.5 μM of Compound A. At various time points, ie 6, 24, 30 hours, cells were harvested or trypsinized and lysed using lysis buffer (Sigma Aldrich, USA). Estimate the protein content. The lysate was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting (Molecular Cancer Therapeutics, 2007, 6, 918-925). Western blotting was performed using specific antibodies to Bcl-2 and actin. The results are described in Figure 4.
As can be seen from Figure 4, Compound A negatively regulates the anti-apoptotic protein Bcl-2 in a dose-dependent manner in both cell lines. In MCF-7 cells, Bcl-2 was significantly negatively regulated from 24 hours, while a significant negative regulation was observed in MDA-MB-231 at 30 hours.


example 8:
Effect of Compound A on Cell Cycle Progression and Apoptosis:
The effect of Compound A and the PARP inhibitor BSI-201 (internal preparation) on cell cycle progression and apoptosis was evaluated in two TNBC cell lines. Non-synchronized human TNBC cell line MDA-MB-231 and MDA-MB-468 with RPMI 1460 with 10% FCS, 0.5 x 10 per bottle⁶The density of cells is planted inTissue culture flasks. After 24 hours, cells were treated with 1.5 and 3.0 μM Compound A or 50 μM PARP inhibitor BSI-201 for 72 hours. Cells were harvested (digested by trypsin) after incubation and as provided in Example 6. The result is shown intable 4A versus 4BAnd presented in graphicsFirst 5A , 5B versus 5C Figure.

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

table 4B :Comparison of percentage distribution of cells with apoptosis at different cell cycle stages in MDA-MB-468 treated with Compound A (CDK inhibitor) and BSI-201 (PARP inhibitor)

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

example 9 :
Effect of Compound A on MCF-7 Cyclin and CDK4 Kinase Activity
Step 1: Basic expression of cyclin D1 performance
Western blot analysis (Molecular Cancer Therapeutics, 2007, 6, 918-925) was used to study across different breast cancer cell lines (ie MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-435 S, MDA) The basic expression of cyclin D1 of -MB-453, BT-549 and HBL-100). These cells were planted in RPMI 1460 medium with 10% FCS.Tissue culture flasks were incubated for 24 hours. Cells will be collected (digested with trypsin) and lysed using lysis buffer. Estimate the protein content. The lysate was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting (Molecular Cancer Therapeutics, 2007, 6, 918-925). The Western blot method was performed using the cyclin D1 antibody and using actin as an injection control group. The results are shown in Figure 6A. High cyclin D1 expression was observed in most breast cancer cell lines including triple negative breast cancer cell lines.

step 2:Effect of Compound A on MCF-7 Cyclin and CDK4 Kinase Activity
MCF-7 cells were seeded in RPMI 1460 medium with 10% FCSTissue culture flasks were incubated for 24 hours. These cells were treated with 1.5 μM Compound A. Cells were harvested (digested by trypsin) at various time points (ie, 3 hours, 6 hours, 9 hours, 12 hours, and 24 hours) and lysed using lysis buffer. The protein content was estimated by the Bradford method (Anal. Biochem., 1976, 72, 248-254). The lysate was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting (Molecular Cancer Therapeutics, 2007, 6, 918-925). Western blotting was performed using specific antibodies to different cyclins (ie, cyclin D1, CDK4, Rb, and pRbSer780).
For immunoprecipitation experiments, MCF-7 cells were synchronized via serum starvation. These cells were treated with 1.5 μM Compound A at different time points (i.e., 3 hours, 6 hours, 9 hours, and 12 hours). Cells were harvested (digested with trypsin) and lysed using lysis buffer and protein content was estimated. CDK4-D1 (cyclin D1 and CDK4) was purified from the lysate by immunoprecipitation using a CDK4-specific antibody. The immune complex was further purified using Protein A Sepharose beads (Sigma Aldrich, USA). Use pRb as the substrate and³²P-labeled ATP (BRIT, India) used immune complexes to determine CDK4 activity. The mixed reactions were used for SDS-PAGE followed by transfer and automated radiography. The results are shown in Figure 6B.
In MCF-7 (Her low, ER+, PR+, BRCA +/- and dual gene loss), Compound A negatively regulates cyclin D1 and pRb in a time-dependent manner. The expression of cyclin D1 and pRb showed a decrease from 6 hours and a significant decrease at 12 hours. Total Rb did not change significantly except for 24 hours. As early as 3 hours, a decrease in CDK4 kinase activity was seen in the cell-based assay.

example 10:
As if by PAR Measured by polymerEffect of Compound A on PARP Enzyme Activity
Poly(ADP-ribose) polymerase (PARP) is a major member of the family of enzymes possessing the catalytic ability of poly(ADP-ribosylation) (PAR). 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% FCSTissue culture flasks were incubated for 24 hours. The cells were treated with 1.5 μM and 5 μM Compound A for 24 hours. Cells were harvested (digested with trypsin) and lysed using lysis buffer. Western blotting is performed using PAR-specific antibodies (Molecular Cancer Therapeutics, 2007, 6, 918-925). The results are shown in Figure 7.
Compound A inhibited PARP enzyme activity as observed by inhibition of PAR polymer formation in the MDA-MB-231 cell line. However, it was observed that the formation of PAR polymer was not inhibited in MDA-MB-468.

example 11:
Effect of Compound A (24 hours) on PARP and cyclin in TNBC cell lines
PARP activity and the correlation of cyclin D1, total Rb and pRb 780 were studied in two TNBC cell lines (ie, MDA-MB-468 and MDA-MB-231). MDA-MB-231 and MDA-MB-468 cells were seeded in RPMI 1460 medium with 10% FCS Tissue culture flasks were incubated for 24 hours. The cells were treated with 1.5 μM and 5 μM Compound A for 24 hours. The cells were harvested (digested with trypsin) and lysed using lysis buffer. Western blotting was performed using PAR, PARP, cyclin D1, CDK4 and pRb Ser 780 specific antibodies (Molecular Cancer Therapeutics, 2007, 6, 918-925). The results are shown in Figure 8.
In MDA-MB-231, Compound A inhibits PARP enzyme activity as seen by inhibition of PAR polymer formation. This is accompanied by a dose-dependent decrease in pRb, cyclin D1 and CDK4. In MDA-MB-468, although there was no change in the formation of PAR polymer, PARP cleavage as an indicator of apoptosis was remarkable.
In the treatment of TNBC cell line MDA-MB-231 with Compound A and culture for 24 hours, inhibition of PARP activity was observed in the cell line. However, MDA-MB-468 did not show that PARP enzyme inhibition instead showed cleavage of PARP. Both of these are markers of cell undergoing apoptosis. Therefore, Compound A is marked to induce significant apoptosis in these cell lines.

example 12:

Test system for the basic gene test of HIF-1α:

1): Genetically engineered cells stably expressing recombinant vectors, wherein the luciferase reporter gene is under the control of three copies of a typical HRE.
2): The control panel cell line comprises a firefly luciferase reporter gene under the control of an essentially active SV40 promoter and enhancer, which helps to exclude luciferase inhibition in a non-specific and/or HIF-1 independent manner. The compound of performance. These cells exhibit high basic performance under normoxic conditions and a slightly lower amount of luciferase under hypoxic conditions.
WillCells were seeded at a volume of 180 μL, 10,000–15,000 cells/well in a 96-well white flat pan at 37 °C.And the environment O₂Cultivate for 24 hours. Compound A was tested at different concentrations (ie, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10 μM) and the flat pan was at 37 ° C., 1 % O₂as well asThe cells were cultured for 20 hours in a modular hypoxia chamber (Billups Rothenberg, MIC 101, USA). After 20 hours of incubation, the tray was removed and allowed to stand at room temperature.And the environmentThe culture was carried out for 1.5 hours. 40 μL of Bright Glo Luciferase Reagent (Promega, USA) was added, and after 3 minutes, fluorescence was measured in a fluorescent mode using a Polar Star Disk Reader (USA). The appropriate control group cells (U251 pGL3) were treated identically except at 37 °C.And the environmentProcess them outside. Compound toxicity was analyzed using the MTS assay. U251 HRE cell line under hypoxiaTreatment with Compound A effectively blocked the performance of HIF-1α in a dose-dependent manner. These compounds are under control of the control cell line under hypoxiaDoes not inhibit luciferase expression. This indicates that Compound A specifically inhibits HIF-1α.
The results are presented graphically in Figure 9.

example 13:
Effect of Compound A on VEGF Inhibition: Cell Line M-9 is a VEGF-Luc construct (VEGF promoter in pGL2-basic) and a geneticin (G418) antibody containing a VEGF promoter reporter gene. The plastids of the sex gene are stably co-transfected with MDA-MB-231. In gene transfer cells, the expression of the reporter gene as measured by luciferase activity is stable.
The effect of Compound A on VEGF inhibition was assessed using a test based on the VEGF reporter gene.
Reagents for the VEGF test:
Lysis Test Buffer (1X)
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 μM ATP-530 μL, 270 μM CoA-1 mL, and 170 μM luciferin - 1 mL.
Luciferase Assay Buffer (LAB) (1X)
Tricin (pH 7.8)-20 mM, magnesium carbonate (Magnesia Alba) - 1.07 mM, MgSO4-2.67 mM, EDTA-0.1 mM, and DTT-33.3 mM.
ATP stock prepared in the laboratory = 5.85 mg/mL
CoA stock prepared in the laboratory = 2.1 mg/mL
Fluorescein stock prepared in the laboratory = 1.5 mg/mL
VEGF Test plan:
InandIn a humidified incubator, M-9 cells were subcultured and maintained in RPMI-1640 medium supplemented with 10% FBS and 4 μL/ml G418 (stock 100 mg/mL).
2. Place the cells in a volume of 180 μL,The density of cells/holes is planted in a tissue culture grade 96-well white plate and a transparent plate, andWetIncubator) Let it attach for 16-20 hours. A total of two sets of discs were made due to different culture conditions.
3. Serially dilute Compound A and BSI-201 in culture medium to achieve the final desired concentration in individual wells (not exceeding 0.5% concentration of DMSO in the wells).
4. Culture conditions: under ambient air conditionsA set of disks is cultured, hereinafter referred to as a normoxic/aerobic disk. The other groups are under anoxic conditions, and their oxygen concentration is less than 1% and 94% nitrogen.This is hereinafter referred to as a hypoxic disk. Culture temperature isAnd the humidity is greater than 75%.
5. After 20-24 hours of incubation under hypoxic and normoxic conditions, the plates were removed from the incubator and the medium removed from all wells of the white plate. The cells were quickly rinsed with 100-150 μL/well of phosphate buffered saline (PBS). Cells were lysed in 40-50 μL of lysis buffer for 20 minutes.
6. Add 100 μL of Luciferase Assay Reagent (LAR) to all wells and immediately place the plate in(Packard, USA) reads the fluorescence. Percent inhibition and inhibitory concentration values () or effective concentration valueIt is calculated by comparing the values of the control group (unprocessed).

VEGF inhibition under hypoxiaValue (μM):
Compound A : 0.31 μM
BSI-201 : > 100 μM
The results are graphically represented in Figure 10.
Treatment with Compound A effectively blocked VEGF expression in a dose-dependent manner.

example 14:
Compound in wound healing test A Effect:
The wound healing test is a simple, inexpensive and one of the earliest developed methods for studying directional cell migration in vitro. This method mimics cell migration during wound healing in vivo.
Protocol: 1. Plant MCF-7 cells in RPMI 1460 medium with 10% FCS.Tissue culture flasks were incubated for 24 hours.
2. Digest the cells with trypsin and per holeThe density is planted in a sterile 6-well plate.
3. Put the plate in a wetIn the incubator)toIncubate for about 16 hours at ambient oxygen levels. The cells are observed to form a dense, uniform monolayer over the entire surface of the pore. The number of cells required for a dense monolayer depends on both the particular cell type and the size of the disc.
4. Use a pipette tip to evenly scrape off the cell monolayer to create a "scratch." The first image of the scratch is captured prior to the addition of the compound.
5. Add Compound A at a concentration of 1 μM and 3 μM.
6. Hold the plate in the incubator for further cultivation. The incubation period is determined empirically by the particular cell type used.
7. After incubation, place the plate under a phase contrast microscope (Zeiss Axio Observer, Germany) to match the reference points, align the photographic area of the first image and capture the second image. The distance between one side of the scratch and the other side was measured for each image.
A similar protocol was followed for BT-549 and MDA-MB-231 cell lines.
The results are shown in Figures 11A, 11B and 11C.
Compound A showed an effective anti-migration effect in all breast cancer cell lines including triple-negative breast cancer cell lines. After 24 hours of culture, the control group cells showed complete healing. Cells treated with Compound A showed minimal migration from both sides, indicating an effective anti-migration effect.

example 15:
Angiogenesis of Compound A in Endothelial Tube Formation Test
The tubular formation test represents a simple but powerful mode for studying the inhibition of vascular proliferation and induction. This test relies on endothelial cells in the extracellular matrix (BD MatrigelTM Matrix),USA) The ability to form thin tubes like the blood vessels, which can then be seen through the microscope. It can be used as a vascular proliferative tubule in a 3-dimensional matrix, which is more similar to a natural physiological environment.
Program
Endothelial tube formation test
Dense HUVECs (human umbilical vein endothelial cells) were cultured to the desired density in the above endothelial medium. A density of 60-80% is recommended for HUVEC.
The endothelial cell suspension was prepared by trypsinizing the cell monolayer and resuspending the cells in the medium with 5-10% serum. Will be every 180 μLThe cell suspension of the cells was added to each well (BD Matrigel Matrix) which was thawed at 4 ° C (each well of a 24-well plate). This suspension was added to the pan and the cultivation continued. The cells were allowed to attach for 2-3 hours and then Compound A (1 mΜ), rotenone (1 μM) (Sigma-Aldrich, USA), and cancer Kangding (3 μM) (Sigma-Aldrich, USA) (20 μL of 10X stock solution) were added. In individual holes. DMSO was used as the control group. After 24-48 hours of incubation, tube formation and vascular proliferation were observed under phase contrast microscopy (Zeiss Axio Observer, Germany).
The results are shown in Figure 12.
Compound 3 effectively inhibits endothelial tube formation and thus inhibits vascular proliferation in a 3D colloid human umbilical vein endothelial cell tube formation assay. Compound A is equivalent to rotenone (standard VEGF inhibitor) at 1 μM and superior to cancer Kangding (HIF-1α inhibitor known in clinical trials).

example 16 :
In vitro cytotoxicity test:
methodPropidium iodide PI Test protocol
The Propidium iodide Fluorescence Test (PI) was carried out according to the procedure described in Anticancer Drugs, 1995, 6, 522-32.
This assay was developed to characterize the in vitro growth characteristics of human tumor cell lines and to test the cytotoxic activity of the test compound. Propidium iodide (PI) is used as a dye that penetrates only the damaged cell membrane. The embedded complex that causes amplification of the fluorescence is generated via PI and double-stranded DNA. After freezing the cells for 24 hours at -20 °C, PI can pass to the whole DNA resulting in a total cell population count. Background readings were obtained from cell-free pores containing medium and propidium iodide.
The human triple-negative breast cancer cell line MDA-MB-231 was planted in a 96-well plate of 180 μL RPMI-1640 medium at a density of 1500-3000 cells/well and moistened.IncubatorThe cells were cultured for about 16 hours to allow the cells to attach. The cells are then processed in two different schedules. In each time frame, use 20 μL of 10X compound in the well (diluted in DMSO and then diluted in cell culture medium to a final DMSO concentration of no more than 0.5%) and plated in a humidified IncubatorUnder cultivation. The medium was removed from the wells and rinsed with PBS. Add 100 μL of PI working solution (7 μg/mL) to each well and store the disk inAbout 16 hours. The plates were thawed and fluorescence was measured using a POLARstar Optical Disk Reader (USA) at excitation 536 nm and emission 590 nm.
(1 mg/mL PI stock solution was prepared by dissolving 1 mg of PI in 1 mL of distilled water. PI working solution was made up to 220 mL (7 μg/mL) by adding 140 μL of the stock solution to PBS. And prepared).

Therapy 1:
Part A: It consists of 4 processing groups.
1) MDA-MB-231 cells were treated with DMSO vehicle and cultured for 24 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added, and cultured for 72 hours (Group IA).
2) Treat the cells in intact medium and incubate for 24 hours, then remove the medium and add compound A.And cultured for 72 hours (Group IIA).
3) GemcitabineCarboplatinThey were treated together and cultured for 24 hours, then the medium was removed, the intact medium was added, and cultured for 72 hours (Group IIIA). (A total of 6 concentrations of continuous double dilution of gemcitabine and carboplatin were performed and the percent inhibition was measured).
4) GemcitabineCarboplatinThey were treated together and cultured for 24 hours, followed by removal of the medium, addition of Compound A, and incubation for 72 hours (Group IVA). (A total of 6 concentrations of continuous double dilution of gemcitabine and carboplatin were performed and the percent inhibition was measured).
The drug treatment schedule is shown in Table 5A.

table 5A.72-hour triple drug combination therapy 1

Part B: It consists of 4 processing groups.
1) Cells were treated with DMSO vehicle and cultured for 24 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 96 hours (Group IB).
2) Treat the cells in intact medium and incubate for 24 hours, then remove the medium and add Compound A ( ) and culture for 96 hours (Group IIB).
3) Gemcitabine Carboplatin They were treated together and cultured for 24 hours, then the medium was removed, the intact medium was added, and cultured for 96 hours (Group IIIB). (A total of 6 concentrations of continuous double dilution of gemcitabine and carboplatin were performed and the percent inhibition was measured).
4) Gemcitabine Carboplatin Treated and incubated for 24 hours, then remove the medium and add compound A And culture for 96 hours (Group IVB). (A total of 6 concentrations of continuous double dilution of gemcitabine and carboplatin were performed and the percent inhibition was measured).
The drug treatment schedule (triple drug combination therapy 1) is shown in Table 5B.
Table 5B. 96-hour triple drug combination therapy 1

The results of Treatment 1 (Parts A and B) are shown in Figures 13A and 13B. Figures 13A and 13B show the effect of different concentrations of gemcitabine and carboplatin in the MDA-MB-231 cell line for 24 hours, followed by Compound A 72 and 96 hours. In the MDA-MB-231 cell line, the combination of gemcitabine and carboplatin was found for 24 hours, followed by Compound A (1.0 μM) of I was synergistic at 72 and 96 hours.
Cytotoxicity assay:

BSI-201, carboplatin, gemcitabine, and Compound A were performed in MDA-MB-231, BT-549, and MDA-MB-468, as described in Table 1A of Example 3, after 48 hours of treatment, by cytotoxicity analysis. MeasuredThe value (μM) is used in Example 16. After completion of compound treatment (i.e., at the end of 48 hours), the discs were subjected to a PI assay and compared to the DMSO (vehicle) control group to calculate percent cytotoxicity. The results indicated that BSI-201 (a PARP inhibitor) showed more than seventy times more than Compound A in all TNBC cell lines.. Compound A showed greater potential in TNBC cell lines as compared to carboplatin and BSI-201 in the anti-proliferation assay.


Therapy 2A: In each of the A, B, C, and D sections, 4 groups are set.
Part A:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 72 hours (Group 1a).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add compound A.And cultured for 72 hours (Group 2a).
3) Treat the cells with gemcitabineAnd cultured for 6 hours, followed by carboplatinThe cells were treated and cultured for 24 hours, then the medium was removed, the intact medium was added, and cultured for 72 hours (Group 3a).
4) Treat the cells with gemcitabine () and cultured for 6 hours, followed by carboplatin () treated and cultured for 24 hours, then the medium was removed and Compound A was addedAnd cultured for 72 hours (Group 4a).
The drug treatment schedule (triple drug combination therapy 2) is shown in Table 6A.

table 6A.Compound AAnd training 72 hours of triple drug combination therapy 2

Part B:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 96 hours (Group 1b).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add compound A.And cultured for 96 hours (Group 2b).
3) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin (The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium and incubation for 96 hours (Group 3b).
4) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin () treated and cultured for 24 hours, followed by further removal of the medium, addition of Compound AAnd cultured for 96 hours (Group 4b).
The drug treatment schedule (triple drug combination therapy 2) is shown in Table 6B.

table 6B.Compound ATreatment of 96-hour triple drug combination therapy 2

Part C:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 72 hours (Group 1a).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add Compound A () and culture for 72 hours (Group 2c).
3) Put the cells in gemcitabine ( Treated and cultured for 6 hours, followed by carboplatin (The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium and incubation for 72 hours (Group 3a).
4) Put the cells in gemcitabine ( Treated and cultured for 6 hours, followed by carboplatin () treated and cultured for 24 hours, followed by further removal of the medium, addition of Compound AAnd cultured for 72 hours (Group 4c).
The drug treatment schedule (triple drug combination therapy 2) is shown in Table 6C.

table 6C :Compound ATreatment of 72 hours of triple drug combination therapy 2

Part D:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 96 hours (Group 1b).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add Compound A () and culture for 96 hours (group 2d).
3) Put the cells in gemcitabine () and cultured for 6 hours, followed by carboplatin (The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium and incubation for 96 hours (Group 3b).
4) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin (Treated and cultured for 24 hours, followed by further removal of the medium, addition of Compound A () and culture for 96 hours (group 4d).
The drug treatment schedule (triple drug combination therapy 2) is shown in Table 6D.

table 6D.Compound ATreatment of 96-hour triple drug combination therapy 2

For the above experiments, the PI cytotoxicity test protocol was used to analyze the dishes at the end of the culture period. The results are shown in Figures 14A and 14B.
Figures 14A and 14B show the effect of binding of gemcitabine to carboplatin to compound A for 72 hours and 96 hours in the MDA-MB-231 cell line. At lower concentrations, gemcitabine and carboplatinAt the end of the 96 hour and 120 hour treatment periods, the respective cytotoxic percentages were 49% and 63.9%. However, when compared with gemcitabine and carboplatin alone, Compound AversusBoth significantly enhanced the cytotoxicity caused by gemcitabine and carboplatin to 60% and 74.5% (at 72 hours) and 77.7% and 88.2% (at 96 hours), respectively. Therefore, the longer the treatment period of Compound A (96 hours) and the higher the concentration of Compound A (ieConcentration) The stronger the cytotoxicity.

Therapy 2B: Comparison of BSI-201 binding studies in MDA-MB-231 cells and binding studies with Compound A
In each of the A, B, C, and D sections, 4 groups are set.
Part A:
1) The cells were treated with DMSO medium and cultured for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added, and cultured for 72 hours (Group 1a*).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add BSI-201And culture for 72 hours (group 2a*).
3) Treat the cells with gemcitabine () and cultured for 6 hours, followed by carboplatin (The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium, and incubation for 72 hours (Group 3a*).
4) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin () treated and cultured for 24 hours, then removed the medium and added BSI-201And culture for 72 hours (Group 4a*). The drug treatment schedule is shown in Table 6A*.

table 6A* :With BSI-201 (=40 μM) treatment of 72 hours of triple drug combination therapy 2

Part B:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 96 hours (Group 1b*).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add BSI-201And culture 96 (group 2b*).
3) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin (The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium, and incubation for 96 hours (Group 3b*).
4) Put the cells in gemcitabine ( Treated and cultured for 6 hours, followed by carboplatin () treated and cultured for 24 hours, followed by further removal of the medium and addition of BSI-201And culture 96 (group 4b*).
The drug treatment schedule is shown in Table 6B*.

table 6B* :With BSI-201 (=40 μM) treatment of 96 hours of triple drug combination therapy 2

Part C:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 72 hours (Group 1a*).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add BSI-201 ( ) and culture for 72 hours (group 2c*).
3) Put the cells in gemcitabine ( ) hours and culture for 6 hours, followed by carboplatin ( The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium, and incubation for 72 hours (Group 3a*).
4) Put the cells in gemcitabine ( Treated and cultured for 6 hours, followed by carboplatin ( ) treated and cultured for 24 hours, followed by further removal of the medium and addition of BSI-201 ( ) and culture for 72 hours (group 4c*).
The drug treatment schedule is shown in Table 6C*.
Form 6C* : with BSI-201 ( =70 μM) treatment of 72 hours of triple drug combination therapy 2

Part D:
1) Cells were treated with DMSO vehicle and incubated for 30 hours, then the medium was removed, intact medium (CM: medium + 10% FCS) was added and cultured for 96 hours (Group 1b*).
2) Treat the cells in intact medium and incubate for 30 hours, then remove the medium and add BSI-201 () and culture for 96 hours (group 2d*).
3) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin (The cells were treated and cultured for 24 hours, followed by further removal of the medium, addition of intact medium, and incubation for 96 hours (Group 3b*).
4) Put the cells in gemcitabine (Treated and cultured for 6 hours, followed by carboplatin () treated and cultured for 24 hours, followed by further removal of the medium and addition of BSI-201 () and culture for 96 hours (group 4d*).
The drug treatment schedule is shown in Table 6D*.

table 6D* :With BSI-201 (=70 μM) treatment of 96 hours of triple drug combination therapy 2

For the above experiments, at the end of the culture period, the PI cytotoxicity test protocol was used to analyze the dishes. The results are shown in Figures 15A and 15B.
Figures 15A and 15B show the effect of binding of gemcitabine to carboplatin and BSI-201 for 72 and 96 hours in the MDA-MB-231 cell line. At lower concentrations (ie gemcitabine and carboplatin)The percentage of cytotoxicity at the end of 96 hours and at the end of 120 hours was 59.7% and 49.9%, respectively.
However, when compared with gemcitabine and carboplatin alone, BSI-201 isversusBoth did not significantly enhance the cytotoxicity caused by gemcitabine and carboplatin (only 62.9% and 63.2% at 72 hours, respectively, and 47.1% and 57.3% at 96 hours, respectively).
The results of the binding studies of BSI-201 in MDA-MB-231 cells and the binding studies by Compound A are presented in Table 7.

table 7 :Percent inhibition of BSI-201 and Compound A binding in MDA-MB-231 cells

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

The synergistic effect in the TNBC MDA-MB-231 cell line has been assessed using the CompuSyn software from Chou and Talalay as described in Pharmacological Reviews, 2006, 58, 621-681. A Combination Index (CI) was used to assess whether the binding is additive, synergistic, or antagonistic. CI < 1 is synergistic, CI = 1 is additive and CI > 1 is antagonistic. The binding index as assessed by the combination of therapy 2A and therapy 2B is shown in Table 8.

Table 8: CI values for the combination of therapy 2A and therapy 2B

The results of the two therapies used in the triple drug binding assay are indicated with:
1) Gemcitabine followed by carboplatin followed by Compound A, and
2) Gemcitabine together with carboplatin followed by compound A
Compound A is synergistic when used in combination.

Example 17:
The effect of compound A and gemcitabine and carboplatin triple drug combination on triple-negative breast cancer xenograft model in SCID mice
OBJECTIVE: The purpose of this study was to evaluate the antitumor activity of Compound A in combination with gemcitabine and carboplatin in a triple negative human breast cancer xenograft model of MDA-MB-231 (mammary adenocarcinoma).
Animal ethics:
The placement and care of animals is based on guidelines effectively published by the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) in Tamil Nadu, India. The procedure for using laboratory animals was approved by the IAEC (Institutional Animal Ethics Committee) of the Research Centre of Piramal Life Sciences Limited.
Method: Human breast cancer MDA-MB-231 cells were cultured in RPMI 1640 medium containing 10% fetal calf serum in a 5% CO ₂ incubator. The cells were pelleted by centrifugation at 1000 rpm for 10 minutes to pellet. Resuspend the cells in a pre-cooled mixture of saline and Matrigel (ratio 1:1) to obtain a count per 0.2 mL solution The cells are kept on ice. This suspension was injected into female SCID mice (5-6 weeks old) via the subcutaneous (sc) route. Mouse tactile tumor masses were observed every other day.
Once the tumor size reached a size of 3-5 mm in diameter, the animals were randomized into individual treatment groups from G1 to G6, including the untreated control group. Compound A, BSI-201, gemcitabine, and carboplatin were administered intraperitoneally as described in Table 9, and tumor measurements were completed every 2-3 days. The percent growth inhibition (GI%) was calculated at the end of the experiment.
Dosing: The dosing schedule for the individual treatment groups G1 to G6 is shown in Table 9. Group allocation and dose (D: day, G: gemcitabine, C: carboplatin)

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

Dosing:
All mice were designed for intraperitoneal administration according to the study design.

Tumor measurement:
a) Calculate the formula for the long ellipsoid in milligrams of tumor weight:

b) For group G4 (tumor treatment with gemcitabine + carboplatin + compound A), the treatment-to-control group ratio (T/C %) on a given day X is calculated using the formula:

among them:
Ax is the tumor size on day X of group G4 (treated with gemcitabine + carboplatin + compound A);
Ao is the tumor size on day 0 of group G4 (treated with gemcitabine + carboplatin + compound A);
Cx is the tumor size of group G6 (treated in the control group) on day X;
Co is the tumor size at day 0 on group G6 (treated in the control group).
The treatment versus group ratios for groups G1, G2, G3, and G5 were calculated identically.

c) growth inhibition (GI) is calculated as

GI % > 75% of the drug is considered very active;
GI % < 50% drug is considered to be inactive.

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




in conclusion:
The binding of Compound A to gemcitabine to carboplatin showed significant in vivo antitumor activity in the triple negative breast cancer xenograft mode of MDA-MB-231.
Compared to BSI-201 and gemcitabine in combination with carboplatin, GI = 82%, Compound A combined with gemcitabine and carboplatin showed anti-tumor activity with GI = 96%. Compound A greatly enhanced the effects of gemcitabine and carboplatin in the conjugate compared to the combination of only gemcitabine and carboplatin or compound A alone.
All treated groups did not show significant weight loss, indicating that the drugs and their combinations were well tolerated.
The invention has been described, it is to be noted that the singular forms "a", "a", "the" and "the" ) includes plural references. Thus, for example, a reference to a composition comprising "a compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally used to include the meaning of "and/or" unless the context clearly dictates otherwise.
All publications and patent applications in this specification are indicative of the extent of
The present invention has been described with reference to various specific and preferred embodiments and techniques. However, it will be appreciated that many variations and modifications can be made within the spirit and scope of the invention.

Bcl-2. . . Anti-apoptotic protein

BSI-201. . . Inhibitor

CDK4. . . Cyclin-dependent kinase-4

ER. . . Estrogen receptor

HER2. . . Human epidermal growth factor receptor 2

HUVEC. . . Human umbilical vein endothelial cells

2a, 2a*, 2b, 2b*, 2c, 2c*, 2d, 2d*, 3a, 3a*, 3b, 3b*, 4a, 4a*, 4b, 4b*, 4c, 4c*, 4d, 4d*, IIIA, IIIB, IVA, IVB. . . Group

MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-435 S, MDA-MB-453, BT-549, HBL-100. . . Cell line

PAR. . . The receptor of PARP enzyme

PARP. . . Poly(ADP-ribose) polymerase

PR. . . Luteinizing hormone receptor

pRb Ser780. . . Phosphorylation-retinblastoma

Rb. . . Retinoblastoma

U251 HRE, U251 pGL3. . . Genetically engineered glioblastoma cells

First1 Figure:Effect of Compound A on Cell Community 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
First 3A Figure:Time-dependent effects of cell cycle progression and apoptosis of compound A in MCF-7 (Her2-, BRCA +/- para-gene loss) cell lines
First 3B Figure:Time-dependent effects of cell cycle progression and apoptosis of compound A in MDA-MB-231 cell line
First 4 Figure:Expression of anti-apoptotic protein Bcl-2 in MCF-7 and MDA-MB-231 cell lines treated with compound A
First 5A Figure:Effect of Compound A on MDA-MB-231 Cell Line (Different Stages of Cell Cycle)
First 5B Figure:Effect of Compound A on MDA-MB-468 Cell Line
First 5C Figure:Effect of BSI-201 on MDA-MB-231 and MDA-MB-468 cell lines
First 6A Figure:Cyclin D1 content in different breast cancer cell lines
First 6B Figure:Effect of Compound A on MCF-7 Cyclin and CDK4 Kinase Activity
First 7 Figure:Effect of Compound A measured by PAR polymer on PARP enzyme activity in breast cancer cell lines (MDA-MB-231 and MDA-MB-468)
First 8 Figure:Effect of Compound A on PARP and Cyclin in MDA-MB-231 and MDA-MB-468 Triple Negative Breast Cancer Cell Lines (24 Hours)
First 9 Figure:Effect of Compound A on HIF-1α Inhibition in U251 HRE and U251 pGL3 Cell Lines
First 10 Figure:Effect of Compound A on VEGF inhibition using a test based on VEGF reporter gene
First 11A Figure: Effect of Compound A on Migration of BT-549 Breast Cancer Cell Line
First 11B Figure:Effect of Compound A on Migration of MDA-MB-231 Breast Cancer Cell Line
First 11C Figure: Effect of Compound A on Migration of MCF-7 Breast Cancer Cell Line
First 12 Figure:Effect of Compound A on the formation of endothelial tubular structures observed in endothelial cell tubular structure formation assay
First 1 3A Figure:The combination of gemcitabine and carboplatin was used in the MDA-MB-231 cell line for 24 hours followed by Compound A () effect for 72 hours
First 1 3B Figure:Different concentrations of the combination of gemcitabine and carboplatin were combined in the MDA-MB-231 cell line for 24 hours followed by Compound A () effect of 96 hours
First 14A Figure:In the MDA-MB-231 cell line, gemcitabine () for 6 hours followed by carboplatin () for 24 hours, followed by compound A (/) effect of a combination of 72 hours
First 14B Figure:In the MDA-MB-231 cell line, gemcitabine () for 6 hours followed by carboplatin () for 24 hours, followed by compound A (/) effect of a combination of 96 hours
First 15A Figure:In the MDA-MB-231 cell line, gemcitabine () for 6 hours followed by carboplatin () for 24 hours, followed by BSI-201 (/) effect of a combination of 72 hours
First 15B Figure:In the MDA-MB-231 cell line, gemcitabine () for 6 hours followed by carboplatin () for 24 hours, followed by BSI-201 (/) effect of a combination of 96 hours
First 16 Figure:Mean tumor growth profile of human breast cancer (MDA-MB-231) xenografts in a triple drug combination study
First 17 Figure:Mean percent growth inhibition of human breast cancer (MDA-MB-231) xenograft model seen in a triple drug combination study
First 18 Figure:Mean percent weight profile of human breast cancer (MDA-MB-231) xenograft model in SCID mice in a triple drug combination study

2a, 2b, 2c, 2d, 3a, 3b, 4a, 4b, 4c, 4d. . . Group

Claims (23)

A pharmaceutical conjugate for use in the treatment of triple-negative breast cancer, wherein the drug conjugate comprises a dicytotoxic anti-tumor agent, gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof, and one of the compounds selected from formula I, CDK An inhibitor or a pharmaceutically acceptable salt thereof;
Wherein Ar is a phenyl group which is unsubstituted or substituted by 1, 2 or 3 identical or different substituents selected from the group consisting of halogens, nitro groups selected from chlorine, bromine, fluorine or iodine. , cyano, C ₁ -C ₄ -alkyl, trifluoromethyl, hydroxy, C ₁ -C ₄ -alkoxy, carboxy, C ₁ -C ₄ -alkoxycarbonyl, CONH ₂ or NR ₁ R ₂ Each of them Independent of the R ₂ system from hydrogen or . The pharmaceutical conjugate as used in claim 1, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the phenyl group is 1, 2 or 3 identical or Substituted by a different substituent selected from the group consisting of halogens selected from chlorine, bromine, fluorine or iodine, Or trifluoromethyl. The pharmaceutical conjugate for use in 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 1, 2 or Three halogens selected from chlorine, bromine, fluorine or iodine are substituted. The pharmaceutical conjugate for use according to any one of 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 Replaced by chlorine. The pharmaceutical conjugate as used in claim 4, wherein the CDK inhibitor is (+)- trans - 2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxyl Methyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (Compound A). The pharmaceutical conjugate for use in 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 a chloro group And a double substitution of a trifluoromethyl group. A pharmaceutical conjugate for use in claim 6 wherein the CDK inhibitor is (+)- trans - 2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8- (2-Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (Compound B). The pharmaceutical conjugate for use according to any one of claims 1 to 7, wherein the cytotoxic antitumor agent gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof and a compound of formula I or The CDK inhibitor represented by the pharmaceutically acceptable salt thereof is continuously administered to a subject in need thereof. The pharmaceutical conjugate as used in claim 8, wherein the cytotoxic antitumor agent gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof is prior to a compound of formula I or a pharmaceutically acceptable compound thereof The CDK inhibitor represented by the salt is administered. A pharmaceutical conjugate for use according to any one of claims 1 to 9, wherein the conjugate exhibits a synergistic effect of treatment. A method for treating a triple negative breast cancer of a subject, comprising: a therapeutically effective amount of a cytotoxic antitumor agent gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount selected from the group consisting of patent applications A CDK inhibitor of the compound of the formula I as defined in the first item or a pharmaceutically acceptable salt thereof is administered to the subject. The method of claim 11, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the phenyl group is 1, 2 or 3 identical or different Substituted by a substituent selected from the group consisting of halogen selected from chlorine, bromine, fluorine or iodine, Or trifluoromethyl. The method of claim 11, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the phenyl group is 1, 2 or 3 Substituted by a halogen selected from chlorine, bromine, fluorine or iodine. The method of any one of clauses 1 to 13, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the phenyl group is Replace. The method of claim 14, wherein the CDK inhibitor is (+)- trans - 2-(2-chloro-phenyl)-5,7-dihydroxy-8-(2-hydroxymethyl) -1-Methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (Compound A). The method of claim 11, wherein the CDK inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the phenyl group is a chlorine group and a The trifluoromethyl group is double substituted. The method of claim 16, wherein the CDK inhibitor is (+)- trans - 2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2) -Hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (Compound B). The method of any one of claims 11 to 17, wherein a therapeutically effective amount of the cytotoxic antitumor agent gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof and a therapeutically effective one are effective The CDK inhibitor of a compound of formula I or a pharmaceutically acceptable salt thereof is administered continuously to the subject in need thereof. The method of claim 18, wherein the therapeutically effective amount of the cytotoxic antitumor agent gemcitabine and carboplatin or a pharmaceutically acceptable salt thereof is preceded by a therapeutically effective amount of Formula I Administration of the CDK inhibitor represented by a compound or a pharmaceutically acceptable salt thereof. The method of any one of clauses 11 to 19, wherein the cytotoxic antitumor agent gemcitabine and carboplatin and the CDK inhibitor exhibit synergistic treatment. The use of a pharmaceutical conjugate as defined in claim 1 of the patent application for the manufacture of a medicament for the treatment of triple-negative breast cancer. The use of the drug conjugate as defined in claim 1 of the patent application, wherein the CDK inhibitor is (+)- trans - 2-(2-chloro-phenyl), as claimed in claim 21, -5,7-Dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-glyoxime-4-one hydrochloride (Compound A). The use of the drug conjugate as defined in claim 1 of the patent application, wherein the CDK inhibitor is (+)- trans- 2-(2-chloro-4-tri) Fluorotolyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-ketone-4-one hydrochloride (Compound B).