TW201242597A - A synergistic pharmaceutical combination for the treatment of pancreatic cancer - Google Patents

Abstract

In accordance with the present invention there is provided a pharmaceutical combination useful for the treatment of pancreatic cancer comprising a CDK inhibitor selected from the compounds of formula I; and a compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity. The present invention also provides a combination wherein said pharmaceutical combination useful for the treatment of pancreatic cancer further comprises gemcitabine. The present invention also provides a method for the treatment of pancreatic cancer in a subject comprising administering said pharmaceutical combination(s) to said subject.

Description

201242597 VI. Description of the Invention: ─ [Technical Field to Which the Invention Is Applicated] [0001] The present invention relates to a pharmaceutical combination for treating pancreatic cancer. The invention particularly relates to a pharmaceutical combination for treating pancreatic cancer comprising a cyclin-dependent kinase inhibitor and a compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity, the cyclin-dependent kinase inhibitor being selected from the group consisting of A compound of Formula I (as described herein) or a pharmaceutically acceptable salt or solvate thereof. The invention more relates to the combination comprising gemcitabine or a salt thereof for use in the treatment of pancreatic cancer. The invention also relates to a pharmaceutical composition comprising the combination; a method for treating pancreatic cancer in an individual, the method comprising administering the pharmaceutical combination to the individual 〇 [Prior Art] [0002] Cancer is a group of characteristics An abnormally controlled disease for cell growth. Cancer cells can invade nearby tissues and 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. Although all types of cancer are pathogenic, pancreatic cancer is the fourth and fifth common cause of cancer-related mortality in North America and Europe, respectively, with an estimated 38,000 new cases per year in the United States and close proximity in Europe. 58, 000 new cases (J. Clin. Oncol.; Volume 27 No. 13, 2231 - 2237 (2009)). In fact, pancreatic cancer has a very poor prognosis with an overall 5-year survival rate of less than 5% 1013264167-0 (Pharmacol Ther 1 9 9 9; 82: 241 - 250). 1{)11.847Bill No. A0101 Page 4 / Total 90 pages 201242597 In addition, patients with advanced 'metastatic and metastatic pancreatic cancer have poor prognosis, and generally too late to diagnose with surgery Or radiation therapy. Chemotherapy provides relief for some patients with advanced pancreatic cancer, but its impact on survival is still not significant so far. Historically, 5-fluorouracil (5-FU) was the drug of choice for systemic therapy for advanced pancreatic cancer, but a single drug study was performed during the period of assessing tumor response with computed tomography (CT). The reaction rate is rarely more than 20%, and the median survival time is 4. 2 - 5. 5 months (J.

Clin. Oncol.; 15:2403 - 241 3 (1 997)). Compared with 2 5-FU, first-line treatment with gemcitabine (Gemzar) produced a modest median survival advantage (5.7 months vs. 4.4 months) and was symptomatic and advanced in randomized clinical trials. The mitigation of patients with the disease appears to be more effective. Although systemic administration of gemcitabine has been associated with clinical benefit and prolonged survival in patients with advanced disease, objective tumor response occurs in less than 10% of patients, and survival time is generally less than 6 months (J. Clin. Oncol.; 1 5:2403 - 2413 (1997)). However, because of the pre-existing chemical resistance or chemical resistance of most tumor cells to drugs, it has been observed during this period that treatment of pancreatic cancer with gemcitabine does not significantly improve the condition of patients with visceral cancer (Oncogene; 22 (21): 3243-51 (2003)). In clinical trials using gemcitabine as a single drug in the treatment of pancreatic cancer, it has been reported that in 2,704 patients, the toxicity and anaerobic toxicity assessed by the World Health Organization are used to assess lung toxicity and allergy toxicity. According to this study, 7.4% of patients had mild symptoms (level 1), and 7.6% of patients experienced dyspnea during activity (grade 2), which occurred in 3.1% of treated patients. Call 1011084#单号纽01 page 5/90 pages 1013264167-0 201242597 Sucking difficulty (level 3) and 〇. 8% of patients need absolute bed rest (special grade 4) (J. pancreas (〇 Niine) 2008. 9(6):708-714). Therefore, treatment with gemcitabine caused anxiety due to the toxic effects reported in patients treated with gemcitabine as monotherapy. Attempts have been made to combine gemcitabine with several other cytotoxic drugs or agents, and from the combination of gemcitabine with cisplatin, 5-FU, Eleno dicken and oxaliplatin in advanced and metastatic pancreatic cancer The results of the study have suggested that the efficacy can be improved. However, Phase III trials of these combinations have not confirmed their advantage over the single agent gemcitabine. In addition, the survival time associated with the combination of gemcitabine/5_FU, gemcitabine/alenodicin, gemcitabine/cisplatin, and gemcitabine/oxaliplatin appears to be equal to or slightly superior to the survival time associated with gemcitabine alone (J. Clin. 〇ncol.: ν〇1. 24 No. 3 327 - 329 (2006)). The combination of gemcitabine and erlotinib (an oral bioavailable guanidine (10) tyrosine inhibitor) shows only additive effects when tested in animal models (J Clin Invest 1 992; 90: 1) 352 - 1 360 ). However, the combination of gemcitabine and erlotinib was used alone in combination with gemcitabine, involving a combination of gemcitabine and erlotinib for the treatment of 569 patients with pancreatic cancer with untreated inoperable surgery. Small but significant survival advantage (〇nc〇1〇gy (Willist〇n

Park) 2003; 17: 11-16). This is the first trial to show a survival advantage for any combination therapy in pancreatic cancer and led to the approval of the Food and Drug Administration (FDA) in 2005 for a combination of front-line therapy for pancreatic cancer. More recently, patients with advanced pancreatic cancer were randomized to receive gemcitabine plus EGFR inhibitor, erlotinib, or only gemcitab 1013264167-0 10110847# single number A01 (n page & total 90 pages) 201242597 Bin's third phase of the 'bed test showed a significant improvement in 6.2 compared to the median survival of 5.9 months and 23% compared to the 17% annual survival rate (

Semin. Oncol· 2005;32:5-6). A clinical trial involving the addition of a second target agent (i.e., bevacizumab) to a combination of gemcitabine and erlotinib has been reported to further improve the outcome of patients with metastatic pancreatic cancer. However, it was observed that the addition of bevacizumab to the gemcitabine _ erlotinib combination did not result in a statistically significant improvement in overall survival in patients with metastatic pancreatic cancer (: 1丨11.〇11(:〇1.; 27: 2231-2237 (2009)). In addition, it has been found that CDK inhibitors, especially yellow-derivatives, synergistically act on gemcitabine in human pancreatic cell lines. A cytotoxic reaction becomes possible (PCT Application Publication No. WO 2008/139271). A combination comprising a VEGF receptor kinase inhibitor and erlotinib or gemcitabine is reported in US Published Patent Application No. US 201 0-0048503, The combination exhibits good anti-tumor activity. Ο Based on the discussion of existing treatment options for pancreatic cancer, especially for locally advanced or metastatic pancreatic cancer, it is clear that no matter what efforts have been made to date, there is still a need to find The new medical approach not only provides improvements in efficacy' but also provides an increased survival advantage for patients suffering from pancreatic cancer. 1011084?#单编号 Further' From the above discussion, it is also clear that although The rules for the combination of anticancer agents with the same mechanism of action may work in some combined examples, and may have no effect on the combination of other anticancer agents in the same way' and this combination may not always result in a combination with beneficial medical effects. However, the invention described herein provides a combination of drugs with improved efficacy and a method for using the combination of drugs in the treatment of pancreatic cancer. The inventors have discovered a combination of known anticancer agents, including 10101. Page 7 of 90 1013264167-0 201242597 Compounds capable of inhibiting EGFR kinase activity and cyclin-dependent kinase inhibitors selected from compounds represented by Formula I (as described herein); when used in pancreatic cancer The treatment provides an unexpectedly superior effect. When used in the treatment of pancreatic cancer, adding the known cytotoxic agent, gemcitabine, to the aforementioned combination further improves the efficacy of the combination [invention] 0003] In one aspect, the invention relates to a pharmaceutical combination comprising a cycle selected from the group consisting of compounds of Formula I a protein-dependent kinase (CDK) inhibitor and a compound capable of inhibiting EGFR kinase activity; for treating pancreatic cancer. In another aspect, the invention relates to a method for treating pancreatic cancer in an individual, comprising administering a medical An effective amount of a cyclin-dependent kinase (CDK) inhibitor is administered to the individual in combination with a therapeutically effective amount of a compound capable of inhibiting EGFR kinase activity, which is selected from the group consisting of compounds of Formula I. In still another aspect, The present invention relates to a pharmaceutical composition comprising a medically effective amount of a cyclin-dependent kinase (CDK) inhibitor selected from the compounds represented by Formula I, and a therapeutically effective amount of a compound capable of inhibiting EGFR kinase activity; for treating pancreatic cancer . In another further aspect, the invention relates to a pharmaceutical combination comprising: a cyclin-dependent kinase (CDK) inhibitor selected from the group consisting of a compound represented by Formula I; and a compound capable of inhibiting EGFR kinase activity; wherein said combination is in the pancreas Synergy is shown in the treatment of cancer.

In still another further aspect, the present invention relates to the pharmaceutical composition comprising a cyclin-dependent kinase selected from the group consisting of the compound of Formula I (CDK 10110847#, single number AQ1Q1 ^ 8 I / * 90 1 1013264167-0 201242597 and capable of inhibiting a compound having EGFR kinase activity; further comprising gemcitabine; for treating pancreatic cancer. In the present invention, the pharmaceutical composition comprises cyclin-dependent kinase (CDK) inhibition in a medically effective compound selected from the formula 1 The agent and the medically effective amount can inhibit the EGFR kinase activity, and further comprise a medically effective amount of gemcitabine; for treating pancreatic cancer. In the case of more visceral cancer, go/close one for use in the individual Treatment of pancreatic fistula [0004] 1011084? #单编号(CDK) inhibition: containing a therapeutically effective amount of a cyclin-dependent enzyme 1 in combination with a therapeutically effective amount of a compound capable of inhibiting EGFR kinase" (4); West He privately administers drugs. / = In another aspect, the present invention relates to a kit, a cyclin-dependent kinase inhibitor of a compound represented by a knife I can inhibit The compound of the EG_enzyme activity is conjugated to the west; the kit may further include a package insert, the challenge 2: no combination treatment is used as a treatment instruction for treatment: The scope of the use of this hair will become obvious.

L-through mode J ^ invention is based in part on CDK inhibitors to enable compounds that inhibit epidermal growth factor receptor (EGFR) kinase activity to become plaque, plaque, enhance the potency of the compound, and produce synergy with it. "Isoto, which is a tolerant peptide of a compound and/or which reduces the side effects caused by the compound, for use in a pancreatic cancer of the family, the CDK inhibitor is selected from the formula VIII of the formula I, and the invention comprises Synergistic 磐遂^ 瞧 page 9 / total 90 ¥ music combination, wherein when using 1013264167-0 201242597 in the treatment of pancreatic cancer, the medical effect of the combination is greater than the addition. Preferably, this combination is also reduced or Avoid unwanted or counter-effects. In certain embodiments, the combinations encompassed by the present invention provide improvements over the administration of a CDK inhibitor or a compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity or gemcitabine. The overall treatment of the present invention. Accordingly, the present invention relates to a pharmaceutical combination designed to treat or control pancreatic cancer in an individual, wherein the combination comprises a CDK inhibitor selected from the group consisting of a compound of formula I and A combination of compounds capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity, and said combination further comprising gemcitabine added thereto. In particular, the present invention provides a method for preventing or controlling pancreatic cancer in an individual, comprising A medically effective amount of a CDK inhibitor selected from a compound of Formula I is administered to the subject in combination with a therapeutically effective amount of a compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity, unless otherwise indicated, the context of the disclosure The general term used hereinbefore and the following general terms preferably have the following meanings. Therefore, the definitions of the general terms used in the context of the present invention are provided below: Unless the context clearly indicates otherwise, the singular form "a (a) "," and "the" include plural references. As used herein, the term "cyclin dependent kinase (CDK) inhibitor" or "CDK inhibitor" means a compound that exhibits activity against one or more known periodic protein-dependent kinases. In the context of the present invention, the CDK inhibitor is a pyrrolidine-substituted flavonoid compound as disclosed in US Application Publication No. 2007001 5802 A1 and PCT Patent Publication No. W02007148158, the entire disclosure of which is incorporated herein by reference. . The CDK inhibitor according to the invention is particularly 10110847^W^A〇101 Page 10 of 90 1013264167-0 201242597 A compound of the formula I or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of the following. Further, as used herein, the term "(10) inhibitor" may mean a compound of formula I and/or a (+)-trans isomer of a formula of a compound of formula IA (as described herein below). And/or a pharmaceutically acceptable salt or solvate of the compound of formula I or a (+)-trans isomer of a compound of formula I. As used herein, the term "a compound capable of inhibiting (10) kinase activity" means a compound which acts by inhibiting, blocking, combating or otherwise blocking EGFR kinase activity in cells and tissues. "Epidermal growth factor bite (EGFR)" is a member of the ErbB family of transmembrane guanine kinase receptors, including sputum (or HER1, or egfr),

ErbB2 (or HER2/neu), ErbB3 (or HER3), and ErbB4 (or HER4). The performance of the test is common in many normal epithelial tissues, and the performance of EGFR is elevated in several solid tumors including pancreatic cancer. As used herein, the term "combination" or "pharmaceutical combination" means a combination of an anticancer agent, ie, a CDK inhibitor, and a compound capable of inhibiting EGFR kinase activity, or an anticancer agent, ie, the CDK inhibitor and A combination of a compound capable of inhibiting EGFR kinase activity and gucitabine; the anticancer agent can be administered separately at a time interval of synergy with the rib partners. The phrase "dual combination" generally means a combination of two known anticancer agents, and comprises a cyclin-dependent kinase inhibitor selected from the compounds represented by Formula I and a compound capable of inhibiting EGFR kinase activity. The term also means a combination administration of an anti-cancer K, a CDK inhibitor, and a compound capable of inhibiting EGFR kinase activity. *Single number 10110847» A0101 Page 11 of 90 1013264167-0 201242597 "A combination of words" means a combination of known anticancer agents comprising gemcitabine, a compound capable of inhibiting EGFR kinase activity, and selected from Formula I A cyclin-dependent kinase inhibitor of the compound represented. The cockroach can also mean a combination administration of an anticancer agent (i.e., a gemcitabine, a (10) inhibitor, and a compound capable of inhibiting EGFR kinase activity). As used herein, the term "synergistic" means that the effects achieved by using the methods and combinations of the invention are greater than the use of anticancer agents (ie, CDK inhibitors, compounds capable of inhibiting EGFR kinase activity, and gemcitabine) as monotherapy. The resulting effect is added. Advantageously, this synergistic effect provides better efficacy at the same dosage and/or prevents or delays the enhancement of multiple drug resistance. By "medically effective amount" for treating pancreatic cancer is meant an amount capable of causing one or more of the following effects in an individual receiving a combination of the invention: inhibiting tumor growth to a certain extent, including, slowing, and complete growth arrest. (ii) reducing the number of tumor cells; (iii) reducing tumor size; (iv) inhibiting (ie, reducing, slowing, or completely stopping) tumor cells infiltrating into peripheral organs; (v) inhibiting (ie, reducing, slowing, or completely (vi) enhances the anti-tumor immune response, which may, but does not necessarily lead to tumor regression or rejection; and/or (vii) alleviate one or more symptoms associated with pancreatic cancer to some extent . The term "individual" as used herein means an animal, preferably a mammal, preferably a human, which requires treatment of pancreatic cancer. In the context of the present invention, the term individual can be used interchangeably with the term patient. As used herein, the term "non-reactive/refractory" is used to describe an individual or patient with pancreatic cancer who has been treated with a cancer therapy currently available to treat pancreatic cancer, such as chemotherapy, radiation therapy, surgery. ,Hol 10110847#单号A0101 Page 12/90 pages 1013264167-0 201242597 Mongolian therapy and/or biological therapy/immunotherapy, where the therapy is not clinically sufficient to treat the patient' makes these patients need additional Effective therapies, such as maintaining insensitivity to therapy. The term can also describe an individual or patient who is responsive to therapy but suffers from side effects, recurrence, development of resistance, and the like. In various embodiments, "unreactive/refractory" means that at least some significant portions of cancer cells are not killed, or their cell division is arrested. In this context, using the term "refractory" accepted in the art, cancer cells can be made in vivo or in test tubes by any method known in the art for analyzing the effectiveness of treatment of cancer cells. It is a "non-responsive/refractory" decision. In various embodiments, the cancer is "non-responsive/refractory" in which the number of cancer cells has not been significantly reduced or increased. As used herein, the phrase "make it possible" means an improvement in the efficacy of a medical agent at its usual or approved dosage. In the context of the present invention, the medical agent is an anticancer agent such as a compound capable of inhibiting EGFR kinase activity or gemcitabine. As used herein, the terms "manage," "managing," and "management" mean that the patient or individual is obtained when administered to a patient or individual. The advantageous effects of the pharmaceutical combination of the present invention are to prevent progression or deterioration of pancreatic cancer. As used herein, the term "overexpression" means excessive expression in a cell, such as a cancer cell, a gene, and/or a protein encoded thereby. A cancer cell that "overexpresses" a protein is a cell that has a significantly higher amount of protein than a non-cancer cell of the same tissue type. The term "dosage form" as used herein shall mean a physical form in which the drug is manufactured and dispensed, such as a lozenge, capsule or injection. 10110847, Single No. A_ Page 13 of 90 1013264167-0 201242597 The term "active ingredient" as used herein shall mean a CDK inhibitor selected from a compound of formula i or a compound capable of inhibiting EGFR kinase activity or gemcitabine. When used in the plural (i.e., active ingredients), the term "active ingredient" shall mean a combination of a CDK inhibitor selected from the compounds of Formula I with a compound capable of inhibiting EGFR kinase activity and gemcitabine. The term "active ingredient" can be used interchangeably with the term "medical agent". The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" means a diluent, adjuvant, excipient or delivery means for administration of a compound of the invention. It has now been discovered that a pharmaceutical combination comprising a CDK inhibitor selected from a compound of Formula I (as described herein) and a compound capable of inhibiting EGFR kinase activity; exhibits synergy when used in the treatment of cancer, particularly pancreatic cancer . In particular, a compound capable of inhibiting EGFR kinase activity when used in combination with a CDK inhibitor of Formula I, when any of erlotinib and a CDK inhibitor of Formula I is used alone to treat pancreatic cancer (e.g., erlotinib) produces a significantly better effect. The inventors have also observed that the synergistic effect of a pharmaceutical combination comprising a CDK inhibitor selected from a compound of formula I (as described herein) and a compound capable of inhibiting EGFR kinase activity is furthermore by means of a known cytotoxic agent Gemcitabine or a pharmaceutically acceptable salt thereof is added to the combination to be enhanced. Accordingly, the invention also includes within its scope a pharmaceutical combination comprising a CDK inhibitor selected from the group consisting of a compound of formula I (as described herein); a compound capable of inhibiting EGFR kinase activity and a gemcitide 1 〇 1 leg #单编号施01 Page 14 / Total 90 pages 1013264167-0 201242597 Bin or its pharmaceutically acceptable salts; for the treatment of pancreatic cancer. The CDK inhibitor used in the pharmaceutical combination of the invention is selected from the compounds of formula I as described herein below. The compound of formula I is a promising CDK inhibitor which inhibits the proliferation of many cancer cells. In a specific embodiment, the CDK inhibitor used in the pharmaceutical combination of the present invention is selected from the compounds represented by the following formula I, [0005]

ΟΗ 〇

Ο wherein Ar is phenyl which is unsubstituted or substituted by 1, 2 or 3 identical or different substituents selected from the group consisting of: i; nitro, cyano, C1-C4-alkyl, Trifluoromethyl, hydroxy or C1-C4-alkoxy; or a pharmaceutically acceptable salt or solvate thereof. In a particular embodiment of the invention, the CDK inhibitor is a (+)-trans isomer of a compound of formula I, as indicated in formula IA below, 10110847#单单A0101 page 15 of 90 pages 1013264167-0 201242597 [0006] OH Ο

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 halogen, nitro, cyano, C1-C4-alkyl, Trifluoromethyl, hydroxy or H-C4-alkoxy; or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the CDK inhibitor used in the pharmaceutical combination of the invention is a compound of formula I wherein the phenyl group is substituted with 1, 2 or 3 identical or different substituents selected from the group consisting of: chlorine, Bromine, fluorine or iodine, C1-C4-alkyl or trifluoromethyl; or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the CM inhibitor used in the pharmaceutical combination of the invention is a compound of formula I wherein the phenyl group is substituted with 1, 2 or 3 halogens selected from the group consisting of chlorine, bromine, fluorine or iodine; A pharmaceutically acceptable salt or solvate thereof. In another embodiment, the CDK inhibitor used in the pharmaceutical combination of the invention is a compound of formula I wherein the phenyl group is substituted with a gas; or a pharmaceutically acceptable salt or solvate thereof. In another embodiment, the CDK inhibitor used in the pharmaceutical combination of the invention is a compound of formula I wherein the phenyl group is substituted with two different substituents 10110847#单号A〇101 Page 16 of 90 pages 1013264167- 0 201242597 Instead, the substituent is selected from the group consisting of chlorophenol trifluoromethyl; or a commercially acceptable salt or solvate. The person skilled in the art (4) appreciates that the CDK inhibitor represented by the molecular formula 含有 compound contains at least two palmar centers, and thus in the form of two different optical isomers (ie (1) or (a) mirror image isomers) presence. All such mirror image isomers as well as mixtures thereof (including racemic mixtures) are included in the present invention. The molecular formula 丨 合 四 四 四 ( ( ( 四 四 pc pc pc pc pc pc pc 卯 卯 卯 卯 卯 卯 卯 卯 卯 卯 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 004 Mirror image isomers of the compounds can also be obtained by methods well known in the art, such as palm HPLC and enzyme riding. Alternatively, the mirror image isomer of the compound of Formula 1 can be synthesized by using an optically active starting material. The manufacture of a compound of the formula I, which may be in the form of a pharmaceutically acceptable salt and a solvate, and the manufacture of an oral and/or parenteral pharmaceutical composition containing the above compound, substantially disclosed in the pCT application Published in the number W02004004632. This patent, which is incorporated herein by reference, discloses that the CM inhibitor represented by the formula 展现 exhibits significant anti-cancer efficacy. As indicated herein above, the CDK inhibitors of Formula I can be used in their salt form. Preferred salts of the formula hydrazine compounds include the hydrochloride, methanesulfonic acid and trifluoroacetate salts. Thus, in another embodiment, the CDK inhibitor used in the pharmaceutical combination of the invention is selected from the group consisting of (+)-trans-2-(2-mono-phenyl)_5,7-dihydroxy-8-(2- Hydroxy-methyl-methyl-pyrrolidinyl-3-yl)-benzophenone or (+)-trans 1013264167-0-an-4-one hydrochloride (referred to herein as compound A) 10110847#单单A0101口页/Total 90 pages 201242597 [2[(2 gas-4 - digas thiol-phenyl)_5, one or two vias a 8_(2_ 'methyl 1 thiopyrobitone-3~) - Benzooxene-4-ketohydrochloride (referred to herein as Compound B). In another further embodiment, the CDK inhibitor used in the pharmaceutical combination of the invention is (+)-trans-2-(2-chloro-phenyl)_5 7_ _hydroxy-8-(2-hydroxyl - mercapto-didecyl-pyrrolidin-3-yl)-benzopipene-4-one hydrochloride (Compound a). In another embodiment, the CM inhibitor used in the pharmaceutical combination of the invention is (+)-trans-3-[2[(2-carb-4-trifluoromethyl-phenyl)-5, 7-Dihydroxy-8-(2-hydroxyindol-1-methyl-pyrrolidine_3_yl)_ stupid bottom 0 hydrochloride (Compound B) ^

According to the present invention, the compound which is capable of inhibiting 阢邝 kinase activity in the pharmaceutical combination of the present invention may be selected from the group consisting of gefitinib (also known as IRESSA® or ZD1 839; AstraZeneca ), erlotinib (also known as TARCEVA® or 0SI-774; 0SI

Pharmaceuticals, Inc.) 'Lapatinib or dimethyl citrate lapatinib (also known as GW57201 6 or TYKERB®; GlaxoSmithKline), AG 1478 (4-(3- aniline) )-6,7-dimethoxy sulfonium sulfonate; AG Technology Co., Ltd.), EKB-569 (peritinib; Wyeth Pharmaceuticals), EKI-785 (Wyeth Pharmaceuticals), PKI_166 (Nova Pharmaceuticals) , Carnitinib dihydrochloride (also known as CI-i〇33; Pfizer), D-69491 (also known as SU11464; Baxter Oncology), Vande Thani (also Known as ZD6474 or ZACTIMA®; AstraZeneca), xl-147 (also known as SAR-245408, Exelixis, Inc.), Alfatini (Bailinger Ingelheim) Company (Boehringer 1013264167-0 then 84# single number A〇101 page 18 / total 90 stomach 201242597

Ingelheim Corp ) \ rilnr ., , .))CUDC~l〇l (Curii

Inc. ) ) x S-22261 1 ru, i (Yan Ye Yi Pharmaceutical Co., Ltd.), AZD-8931 (Astjie inch Tillicom) or neratinib (Wyeth Pharmaceuticals) ° specific to this month In the examples, the compound which inhibits EGFR kinase activity which can be used in the pharmaceutical combination of the present invention is selected from erlotinib or lapatinib or a pharmaceutically acceptable salt thereof. : Rotithini is an EGFR inhibitor. This drug is modeled after gefitinib (Iressa p. The first drug for this type of fentanyl. Erlotinib-specific soil also records epidermal growth factor receptor (EGFR) glutamate kinase, which is Various forms of cancerous towels are highly expressed and sometimes mutated. They bind reversibly to the receptor's triplate acid (4) p) binding site (J Clin Oncol 2007; 25: 1960-1966). Erlotinib is commercially available. Lapatinib is a 4-anilinoinline derivative capable of reversibly inhibiting EGFR and visceral 2(10) aminase activity. Like other small molecule carbamide-inhibitors, lapatinib competes with ATp for its binding position on the glutamate kinase region. In the cell-free biochemical kinase assay, lapatinib inhibited recombinant EGFR and HER2 tyrosine kinase by 50% (IC50) at a frequency of 10.8 and 9.3 nmol/1. It acts as a reversible inhibitor with an estimated dissociation constant (Ki) of 3 and 13 nm 〇l/l for EGFR and HER2, respectively [Drugs6() Suppl 1: i5-23]. Lapatinib is commercially available. 1013264167-0 Gemcitabine is the scientific name designated as 2,-deoxy-2,,2,-difluorocytosine. It is commercially available as a monohydrochloride as well as a chiral isomer. Gemcitabine is also known as Gemcitabine® and is a cytotoxic agent. It is a kind of cause of 1011084# item number deletion 1 帛 19 pages / total 9 pages 201242597 DNA synthesis is inhibited by antimetabolites. Gemcitabine is disclosed in U.S. Patent Nos. 4,808,614 and 5,464,826, the disclosure of which is incorporated herein by reference in its entirety for the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure. A commercial formulation of gemcitabine hydrochloride as a single agent has been identified as a first line treatment for patients with locally advanced or metastatic adenocarcinoma of the pancreas. Gemcitabine is a commercially available oxime. In one embodiment, the invention relates to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises a cycle selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof A protein-dependent kinase (CM) inhibitor; a compound selected from erlotinib or lapatinib and capable of inhibiting EGFR kinase activity or a pharmaceutically acceptable salt thereof. In another embodiment, the invention is directed to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises a cyclin-dependent kinase (CDK) selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof An inhibitor and erlotinib or a pharmaceutically acceptable salt thereof. In another embodiment, the invention is directed to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises a cyclin-dependent kinase (CDK) selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof An inhibitor and lapatinib or a pharmaceutically acceptable salt thereof. In another embodiment, the invention is directed to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises a CDK inhibitor selected from Compound A or Compound B and is selected from the group consisting of erlotinib or lapatinib. A compound capable of inhibiting EGFR kinase activity or a pharmaceutically acceptable salt thereof. In another embodiment, the invention is directed to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises Compound A and erlotinib or 1〇11〇847#单单施〇1 1013264167-0 20th Page / Total 90 pages 201242597 Its pharmaceutically acceptable salts. In another embodiment, the pharmaceutical combination of the present invention, wherein the combination comprises a medicinal salt for treating sputum. . In the partial and lapatinib or in the embodiments, the present invention comprises within its scope a CDK inhibitor of gemcitabine and a compound of the formula (4) of the formula 1 as being selected from erlotinib or sirolimus or its pharmaceutically acceptable = A pharmaceutical combination of compounds that are kinase active. Therefore, in It ° contains a compound selected from the formula 二 : : : : : : : : : : : : :

呶m (d) Ninilapatinib and a compound capable of inhibiting EGFR or a pharmaceutically acceptable salt thereof; and gemcitabine. » = In the medical application, the present invention is directed to a medical music combination for treating post-operative cancer, wherein the combination comprises

= _ or solvate avoidance; two substances: (the (four) or its pharmaceutically acceptable (four) and Kyrgyzstan in another specific example, this I cancer _ combination, where · bis::; - dirty: a cyclin-dependent kinase of a dimer: (10) a nicotinic acid or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof. In a specific embodiment, the invention is directed to

= combination, wherein the combination comprises a compound selected from the group consisting of compound (IV) B #单单1(四)_ (m) inhibiting her 34 erlotinib or

1011084#单单除1 * 21 I / go I 1013264167-0 201242597 Lapatinib and a compound capable of inhibiting EGFR kinase activity or a pharmaceutically acceptable salt thereof; and gemcitabine or a pharmaceutically acceptable salt thereof. In another embodiment, the invention is directed to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises Compound A; erlotinib or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof In another embodiment, the invention is directed to a pharmaceutical combination for treating pancreatic cancer, wherein the combination comprises Compound A; lapatinib or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable thereof a salt of hydrazine, in a particular embodiment, comprising a pharmaceutical combination selected from the group consisting of a compound of formula I and a CDK inhibitor of erlotinib or lapatinib, or comprising the CDK inhibitor; erlotinib or lapata a combination of nicotine and gemcitabine; not specifically limited to those obtained by physical association of the components, but also those that allow for separate, continuous or intermittent administration over a period of time to obtain the combination Maximum efficiency. Thus, the pharmaceutical combination can be administered simultaneously or over a period of time to effectively treat pancreatic cancer. In a specific embodiment, the present invention relates to a pharmaceutical composition comprising a CDK inhibitor selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt or solvate thereof, and a selected from erlotinib or pull Patinib and a compound capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier. In another embodiment, the invention relates to a pharmaceutical composition comprising a CDK inhibitor selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, and erlotinib or a pharmaceutically acceptable thereof 1 〇 47 47# 单编号 删除1 Page 22 / Total 90 pages 1013264167-0 201242597 Salts to be accepted; combined with pharmaceutically acceptable excipients or carriers. In another further embodiment, the invention relates to a pharmaceutical composition comprising a CDK inhibitor selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, and lapatinib or A pharmaceutically acceptable salt; in combination with a pharmaceutically acceptable excipient or carrier. In another further embodiment, the invention relates to a pharmaceutical composition comprising a compound A or compound B a CDK 0 inhibitor and a compound selected from erlotinib or lapatinib capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier. In another further embodiment, the invention relates to a pharmaceutical group. The pharmaceutical composition comprises Compound A and erlotinib or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier, in another further embodiment, the invention relates to a Medicinal Q group, the pharmaceutical composition comprising Compound A and lapatinib or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier, according to a particular embodiment, the invention is A pharmaceutical composition comprising (10) an inhibitor selected from the group consisting of the compound of the formula I or a pharmaceutically acceptable salt or solvate thereof, and a erectinib or lapatinib capable of inhibiting EGFR kinase activity a compound or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier; the composition further comprises gemcitabine or a pharmaceutically acceptable salt thereof, in which the invention relates to a pharmaceutical composition 10110847# Number 1 is page 23 / total 90 pages. Accordingly, the pharmaceutical composition package 1013264167-0 201242597 contains a CDK inhibitor selected from the compound of the formula i or a pharmaceutically acceptable salt or solvate thereof; the ability selected from erlotinib or lapatinib a compound that inhibits EGFR kinase activity, or a pharmaceutically acceptable salt thereof; and gemcitabine; in combination with a pharmaceutically acceptable excipient or carrier. In another embodiment, the invention relates to a pharmaceutical composition comprising a CDK inhibitor selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof; erlotinib or a pharmaceutically acceptable Accepted salts and gemcitabine or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier. In another further embodiment, the invention relates to a pharmaceutical composition comprising a CDK inhibitor selected from the group consisting of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof; lapatinib or A pharmaceutically acceptable salt; and gemcitabine or a pharmaceutically acceptable salt thereof; in combination with a pharmaceutically acceptable excipient or carrier. In another embodiment, the present invention relates to a pharmaceutical composition comprising a CDK inhibitor selected from the group consisting of Compound A or Compound B; and for inhibiting EGFR kinase selected from erlotinib or lapatinib An active compound or a pharmaceutically acceptable salt thereof; and gemcitabine or a pharmaceutically acceptable salt thereof; in association with a pharmaceutically acceptable excipient or carrier. In another further embodiment, the invention relates to a pharmaceutical composition comprising Compound A; erlotinib or a pharmaceutically acceptable salt thereof; and gemcitabine or a pharmaceutically acceptable salt thereof; In combination with a pharmaceutically acceptable excipient or carrier. In another further embodiment, the invention relates to a pharmaceutical composition comprising Compound A; lapatinib or a pharmaceutically acceptable salt thereof; and gemcitabine or a pharmaceutically acceptable salt thereof; L〇n0847bill number A0101 Page 24/90 pages 1013264167-0 201242597 in combination with a pharmaceutically acceptable excipient or carrier. According to a particular embodiment, the invention is directed to a method for treating septic cancer in an individual comprising a medically effective amount selected from the formula! a CDK inhibitor of a compound or a pharmaceutically acceptable salt thereof; and a medically effective amount of a compound selected from erlotinib or lapatinib capable of inhibiting EGFR kinase activity or a pharmaceutically acceptable salt thereof, is administered to said An individual; wherein the CDK inhibitor and the compound capable of inhibiting EGFR kinase activity are administered simultaneously or sequentially. In another embodiment, the invention is directed to a method for treating (7) pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from the group consisting of a compound of the formula or a pharmaceutically acceptable salt thereof And a medically effective amount of erlotinib or a pharmaceutically acceptable salt thereof is administered to the individual: wherein the CDK inhibitor and erlotinib are administered simultaneously or sequentially. In another further embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt thereof, The drug is administered to the subject in a pharmaceutically effective amount of lapatinib or a pharmaceutically acceptable salt thereof, wherein the CDK inhibitor and lapatinib are administered simultaneously or sequentially. In another further embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising comprising a medically effective amount of a CDK inhibitor selected from Compound A or Compound B and a medically effective amount selected from the group consisting of A compound capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof, of erlotinib or lapatinib is administered to the subject; wherein the compound #A or compound B and erlotinib are administered simultaneously or sequentially . In another further embodiment, the present invention is directed to a method for treating pancreatic cancer in an individual, comprising a therapeutically effective amount of Compound A, and a method for use in the treatment of pancreatic cancer in an individual, 10110847, single number, page 25, page 90, 1013264167-0 201242597 A medically effective amount of erlotinib or a pharmaceutically acceptable salt thereof is administered to the subject; wherein the Compound A and erlotinib are administered simultaneously or sequentially. In another further embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising administering a therapeutically effective amount of Compound A and a medically effective amount of lapatinib or a pharmaceutically acceptable salt thereof Administration to the individual; wherein the Compound A and lapatinib are administered simultaneously or sequentially. In another embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising administering a therapeutically effective amount of the Compound A and a medically effective amount of erlotinib or a pharmaceutically acceptable salt thereof Administration to the subject; wherein the Compound A and erlotinib are administered sequentially such that the Compound A is administered before or after erlotinib. In another further embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising administering a therapeutically effective amount of Compound A and a medically effective amount of lapatinib or a pharmaceutically acceptable salt thereof The drug is administered to the individual; wherein Compound A and lapatinib are administered sequentially such that Compound A is administered before or after lapatinib. According to a particular embodiment of the invention, the method for treating pancreatic cancer in an individual; in addition to a medically effective amount of a CDK inhibitor selected from a compound of formula I or a pharmaceutically acceptable salt thereof, and a medically effective amount a compound selected from erlotinib or lapatinib and capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof, is administered to the individual; further comprising a medically effective amount of gemcitabine or a pharmaceutically acceptable thereof The salt is administered to the individual. Thus, in this particular embodiment, the invention is also directed to a method for treating pancreatic cancer in an individual, comprising a method for treating a medically effective amount, in a 10110847^^'« A0101 page 26/90 page 1013264167-0 201242597 A CDK inhibitor of a compound of Formula I, or a pharmaceutically acceptable salt thereof, is a therapeutically effective amount of a compound selected from erlotinib or lapatinib and capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof; A medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof is administered to the subject; wherein the CDK inhibitor, the compound capable of inhibiting EGFR kinase activity, and gemcitabine are administered simultaneously or sequentially. In another embodiment, the invention relates to a method for treating pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt thereof; An amount of a compound selected from erlotinib or lapatinib and capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof, administered to said subject; Among them, the CDK inhibitor, the compound capable of inhibiting EGFR kinase activity, and gemcitabine are administered simultaneously. In another embodiment, the invention relates to a method for treating pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from the group consisting of a compound of Formula I or a pharmaceutically acceptable salt thereof; An amount of a compound selected from erlotinib or lapatinib and capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof, administered to said subject; Wherein the CDK inhibitor; the compound capable of inhibiting EGFR kinase activity and gemcitabine are administered sequentially such that gemcitabine is administered before or after administration of the CDK inhibitor and the compound capable of inhibiting EGFR kinase activity. According to another specific embodiment, the present invention is directed to a method for treating pancreatic cancer in an individual, comprising a medically effective amount selected from the group consisting of 10 Li 4#单单纲 01 page 27 / 90 pages 1013264167-0 201242597 a CDK inhibitor of a compound of formula i or a pharmaceutically acceptable salt thereof; a medically effective amount of erlotinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof Said individual; wherein the CDK inhibitor, erlotinib and gemcitabine are administered simultaneously or sequentially. According to another specific embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from a compound of Formula I or a pharmaceutically acceptable salt thereof; a pharmaceutically acceptable salt of lapatinib or erlotinib; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof; wherein the CDK inhibitor, lapatinib, and gemcitabine Being administered simultaneously or sequentially. According to another specific embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from a compound of Formula I or a pharmaceutically acceptable salt thereof; Erlotinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof; wherein the CDK inhibitor, erlotinib, and gemcitabine are administered simultaneously. According to another specific embodiment, the invention is directed to a method for treating pancreatic cancer in an individual comprising a medically effective amount of a CDK inhibitor selected from a compound of Formula I or a pharmaceutically acceptable salt thereof; Erlotinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof; wherein the CDK inhibitor, erlotinib, and gemcitabine are administered sequentially, Gemcitabine was administered before or after administration of the CDK inhibitor and erlotinib. Xie No. #单号谢01 1013264167-0 Page 28 of 90 201242597 According to another specific embodiment, the present invention is directed to a method for treating pancreatic cancer in an individual 'comprising a medically effective amount selected from the formula "(1) a compound or a pharmaceutically acceptable salt thereof (inhibitor; a medically effective amount of lapatinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof The salt is administered to the individual; wherein the CDK inhibitor, lapatinib, and gemcitabine are administered sequentially such that gemcitabine is administered before or after administration of the CDK inhibitor and lapatinib. According to another embodiment The invention relates to a method for treating pancreatic cancer in an individual comprising inhibiting a medically effective amount of CDK selected from Compound A or Compound B; a medically effective amount selected from the group consisting of erlotinib or lapata a compound capable of inhibiting EGFR kinase activity, or a pharmaceutically acceptable salt thereof, and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof, administered to said individual; wherein said (10) The preparation, the compound capable of inhibiting EGFR kinase activity, and gemcitabine are administered simultaneously or sequentially. ❹ According to another specific embodiment, the method for treating pancreatic cancer in an individual includes a medically effective amount. Compound A; medically, in an amount of erlotinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of a ® or a pharmaceutically acceptable salt thereof to the individual; wherein the compound A, Erlotinib and gemcitabine are administered simultaneously or sequentially. According to another embodiment, the present invention relates to a method of treating smear cancer in an individual comprising a therapeutically effective amount of Compound A; a therapeutically effective amount Erlotinib or a pharmaceutically acceptable salt thereof; and a medically acceptable dose of gemcitabine which is rarely acceptable for administration to the individual 10101 帛 29 pages / 90 pages 1013264167-0 201242597: wherein Compound A, erlotinib, and gemcitabine are administered simultaneously. According to another embodiment, the invention relates to a method for treating pancreatic cancer in an individual, comprising Amount of Compound A; a medically effective amount of erlotinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof, administered to the individual: wherein Compound A, Ero Dini and gemcitabine are administered sequentially such that gemcitabine is administered before or after administration of Compound A and erlotinib. According to another embodiment, the present invention relates to a method for treating pancreatic cancer in an individual, comprising A medically effective amount of Compound A; a medically effective amount of lapatinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof, administered to said subject; wherein said Compound A, Lapatinib and gemcitabine are administered simultaneously or sequentially. According to another specific embodiment, the invention relates to a method for treating pancreatic cancer in an individual comprising a therapeutically effective amount of Compound A; a medically effective amount of lapatinib or a pharmaceutically acceptable salt thereof; A medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof is administered to the subject; wherein the compound A, lapatinib, and gemcitabine are administered simultaneously. According to another embodiment, the invention relates to an individual A method for treating pancreatic cancer comprising administering a therapeutically effective amount of Compound A; a medically effective amount of lapatinib or a pharmaceutically acceptable salt thereof; and a medically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof to The individual: wherein the compound A, lapatinib and gemcitabine are administered sequentially 1〇1 brain# single number should be 01 1013264167-0 page 30 / total 90 pages 201242597, making gemcitabine in the administration of compound A and Lapa Nitrogen is administered before or after. Administration of a triple combination consisting of a CDK inhibitor of Formula I, a compound selected from erlotinib or lapatinib and capable of inhibiting EGFR kinase activity, and gemcitabine can produce a CM inhibitor of the formula I, Ero The fruiting effect achieved by any of the fentanyl/lapatinib and gemcitabine is better than the combination of the CDK inhibitor of the formula I and the erlotinib or the CDK inhibitor of the formula I and lapatinib. Good results, such as anti-cancer effects. In a particular embodiment, the ingredients included in the combination may have to be administered in different routes due to their different physical and chemical properties. For example, 'a CDK inhibitor of Formula I can be administered orally or parenterally to produce and maintain a good gold content' while gemcitabine and a compound capable of inhibiting EGFR kinase can be administered orally or parenterally, by vein Administration by internal, subcutaneous or intramuscular route.

G. Combinations and compositions intended for pharmaceutical use can be prepared according to any method known in the art for making pharmaceutical combinations and compositions, for example, Remington - The Science and Practice of Pharmacy (21st Edition) (2005) 'Goodman &

Gilman's The Pharmacological Basis of Therapeutics (11th Edition) (2006) and Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (9th Edition) (2011) edited by Allen et al., Lippincott Williams & ffilkins,

Solid-State Chemistry of Drugs (2nd Edition) (1999), each of which is incorporated herein by reference. *单煸10110847^ A0101 Page 31 / Total 90 pages 1013264167-0 201242597 The compositions described herein may be in a form suitable for oral administration, such as a lozenge or capsule, for nasal administration or by inhalation. Administration, for example as a powder or solution, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example as a sterile solution, suspension or emulsion, for topical administration, for example as an ointment or Creams for rectal administration, such as suppositories, or by route of administration by direct injection into a tumor or by regional delivery or by topical delivery. For oral use, the CDK inhibitor of Formula I can be administered, for example, in the form of a troche or capsule, a powder, a dispersible granule or capsule, or as an aqueous solution or suspension. In the case of lozenges for oral use, carriers which are commonly used include lactose, corn starch, magnesium carbonate, talc, and sugar, and a lubricant such as magnesium stearate is often added. For oral administration in a capsule form, useful carriers include lactose, corn starch, magnesium carbonate, talc, and sugar. For intramuscular, intraperitoneal, subcutaneous, and intravenous use, sterile solutions of the active ingredient (such as a CDK inhibitor described herein or a compound capable of inhibiting EGFR kinase activity or gemcitabine) are often employed, and the pH of the solution should be It is properly adjusted and buffered. In a preferred embodiment, the medical agent contained in the combination of the present invention is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to a conventional procedure. Typically, the medical agent contained in the combination of the invention for intravenous administration is a solution in sterile isotonic buffer. The composition may also include a co-solvent when necessary. Compositions for intravenous administration may optionally include a local anesthetic, such as lignocaine, to alleviate pain at the site of the injection. In general, ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry powder in a sealed container or 101 brain 7 number A A 101 page 32 / 90 pages 1013264167-0 201242597 A concentrate, such as an ampoule or pouch that indicates the amount of active agent. When one or more of the medical agents contained in the combination of the invention is administered by infusion, it can be administered, for example, using an infusion bottle containing sterile pharmaceutical grade water or saline. When the medical agent contained in the combination of the present invention is administered by injection, an ampoule of sterile water or saline for injection can be provided to allow the ingredient to be mixed prior to administration. Compositions for oral delivery can be in the form of lozenges, diamond shaped ingots, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Orally administered compositions may contain one or more optional agents, for example, sweeteners, such as sugar, aspartame or saccharin; flavoring agents such as mint, wintergreen oil or cherries; colorants; and preservatives An agent to provide a pharmaceutically elegant preparation. Furthermore, in the form of a lozenge or pill, the composition can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selective penetrating membranes surrounding the osmotically active actuating compound are also suitable for use in oral administration compounds or anticancer agents which are included in the combinations of the invention. In these latter platforms, fluid from the environment adjacent to the capsule is absorbed by the driving compound (anticancer agent) which expands to replace the agent or composition via the pores. These shipping platforms provide the features of the necessary zero-level shipping, as opposed to the spike characteristics of the immediate release formulation. Time delay materials such as glyceryl monostearate or glyceryl stearate can also be used. Oral compositions can include standard delivery vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. This delivery tool is preferably a pharmaceutical grade. Further, the effect of the compound (anticancer agent) contained in the pharmaceutical composition of the invention can be delayed or prolonged by an appropriate formulation. For example, the slow-soluble small particles of the compound No. A0101, page 33/90, 1013264167-0 201242597, can be prepared and incorporated into a tablet or capsule. This technique can be improved by making small particles of several different dissolution rates and filling the capsules with a mixture of such small particles. Tablets or capsules can be coated with a film that is resistant to dissolution for a predictable period of time. Even non-oral preparations can be made long-lasting by dissolving or suspending the compound (anticancer agent) in an oily or emulsified delivery tool that allows it to be slowly dispersed in serum, by the same or different Routes and Compounds capable of inhibiting EGFR kinase activity in a wide variety of different dosage forms, when used in a dual combination, may be combined with a CM inhibitor of Formula I or when used in a triple combination with a CDK inhibitor of Formula I And gemcitabine is administered together or separately. For example, a compound capable of inhibiting EGFR kinase activity is preferably administered orally or parenterally, wherein when the compound capable of inhibiting EGFR kinase activity is erlotinib HC1 (TARCEVA®), oral administration is preferred, and EGFR kinase can be inhibited. The active compound can be used as a tablet, capsule, diamond, throat, hard candy, powder, spray, cream, ointment, suppository, jelly, gel, cream, lotion, ointment, tincture, syrup And various pharmaceutically acceptable blunt carriers such as such forms are administered. Administration of such dosage forms is carried out in single or multiple doses. Carriers include solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents, and the like. Oral pharmaceutical compositions can be suitably sweetened and/or flavored. In another embodiment, the invention is directed to a method for treating pancreatic cancer, the method comprising administering a medically effective amount of the combination to an individual in need of such treatment. Thus, in the method of the present invention, a medical amount of gemcitabine for treating cancer and a medically effective amount of a compound selected from the formula I or a pharmaceutically thereof thereof by 10110847#single number A0101 page 34/90 pages 1013264167-0 201242597 A CDK inhibitor that accepts a salt or solvate and a combination of compounds capable of inhibiting EGFR kinase activity can be administered to an individual in which a synergistic effect is produced to treat a smear cancer in the individual. When used in combination, a synergistic effect between a 'medical agent (anticancer agent), a CDK inhibitor selected from a compound of formula I, a compound capable of inhibiting EGFR kinase activity (eg, erlotinib or lapatinib, and gemcitabine) Analysis was performed in selected pancreatic cell lines. Substantially as described in the experimental section, standard cytotoxicity assays were performed in this cell line using a fixed or altered ratio of therapeutic agents when used in combination with any of the medical agents alone. law. The results were analyzed using the combination index (CI) method, by which a CI value of less than 1 indicates synergy, a value of 1 indicates an additive effect, and a value greater than 1 indicates an antagonistic effect. The present invention encompasses a method for treating or controlling pancreatic cancer in a patient comprising administering an inhibitor comprising a compound of formula I with a compound capable of inhibiting EGFR kinase and further a combination with gemcitabine to the patient. The term "combination" is not limited to administration of a medical agent (anticancer agent) at exactly the same time, and more preferably means that a CDK inhibitor selected from the compound of the formula I and other medical agents are sequentially administered and administered to the individual at a time interval. This allows the CDK inhibitor to act with the other medical agents to provide more benefits than if they were administered in another way. For example, each medical agent can be administered sequentially in any order at the same time or at different time points; however, if they are not administered at the same time, they should be administered in close enough time to provide the desired medical benefit. . Each medical agent can be administered separately in any suitable form and by any suitable route of administration, 1013264167-0 10110847#, single number A0101, page 35/90, 201242597. In a specific embodiment of the invention, the anticancer agent contained in the combination can be administered in a staggered therapy, i.e., the CM inhibitor is administered during a cycle of treatment at a different time than the compound capable of inhibiting EGFR kinase activity and gemcitabine. The time difference of administration of at least two anticancer agents may range from several minutes, hours, days, weeks or longer. Thus, the term combination (or combination) does not necessarily mean that the drug is administered at the same time or as a single dose or a single composition, but each component is administered during the desired treatment period. Anticancer agents can also be administered by different routes. In one embodiment, one "cycle" includes 21 days. These therapies or cycles can be repeated or altered as desired. Other dose therapies and changes are predictable' and are determined by the doctor's guidance. In a particular embodiment, the medical agent is administered during the period in which the two agents are still active. Those skilled in the art will be able to determine such time periods by determining the half-life of the administered medical agent. As indicated previously herein, the active ingredients contained in the medicinal composition can be administered simultaneously or sequentially. For the effective administration of the medical agent contained in the pharmaceutical composition of the present invention, especially when a dual combination is used, including erlotinib in a general dosage range of 25 mg to 3 mg; and at 100 rag/m2/day A CDK inhibitor of Formula I, such as Compound A, is administered in a general dosage range of 185 mg/m2/day. For the effective administration of a medical agent contained in the pharmaceutical combination of the present invention, particularly when a double combination is used, including erlotinib in a general dosage range of 25 fflg to 3 〇〇mg; and at 1 〇〇 mg/ For example, compound A, for a CDK inhibitor of formula I, in the general dosage range of ra2/day to 2〇〇mg/m2/day, 1〇11〇847, single order number A0101, page 36/total 9 page 1013264167-0 201242597 And gemcitabine is administered in a general dosage range from 200rag/m2 dose to 1 000rag/m2 dose. Those skilled in the art will recognize that several variations are possible within the scope and spirit of the invention. The invention will be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the invention, but should not be construed as limiting its scope in any way. Example 1 : A) General procedure for the preparation of a CDK inhibitor molecule of the formula I: The compound of formula I can be prepared according to the method disclosed in PCT Patent Publication No. WO2004004632 and PCT Patent Publication No. WO 020781-8, which This is incorporated herein by reference. A general procedure for the preparation of a compound of formula I or a pharmaceutically acceptable salt thereof comprises the steps of: (a) treating the mirror image isomerism of an intermediate compound of formula VIA using acetic anhydride in the presence of a Lewis acid catalyst Pure (-)-trans mirror image isomer,

To obtain an analytical formula of the compound of formula VIIA, 10Π0847#单号舰01第37页/90 pages 1013264167-0 3201242597 [0008]

OCH

CH3O

3COCH OH, CH2OCOCH3 CH3

VIIA (b) In the presence of a solvent and a solvent, the resolved acetylated compound of the formula VIIA is reacted with an acid of the formula ArCOOH or an acid hydride of the formula ArCOCl or an anhydride of the formula (ArC0) 90 or an ester of the formula ArC00CHq. Wherein Ar is a compound of formula VIIIA, as defined above for a compound of formula I, to obtain an analytical formula;

Ch3o

VEHA (c) treating the resolved compound of formula VI11A with a base in a suitable solvent to obtain the corresponding resolved b-diketone compound of formula IXA; 101 s#####1313264167-0 Page 38 of 90 CH3, 〇0 ◦

Ch3 201242597 [0009] wherein Ar is as defined above; Ο [0010] (d) treating the resolved b-diketone compound of formula IXA with an acid (eg, hydrogen acid) to obtain the corresponding cyclized compound of formula XA,

XA

(e) Dealkylation of a compound of formula XA by heating a compound of formula XA with a dealkylating agent at a temperature in the range of from 1 20 to 80 ° C to obtain a (+)-trans mirror of the compound of formula I The isomer and, optionally, the title compound is converted to its pharmaceutically acceptable salt. The Lewis acid catalyst used in the above step (a) may be selected from the group consisting of BF3, E12 0, zinc hydride, chlorinated iron, and titanium carbide. The base used in the process step (b) may be selected from the group consisting of triethylamine, pyridine and a combination of DCC-DMAP (a combination of hydrazine, Ν'-dicyclohexyldiimide and 4-dimercaptoaminopyridine). HHIOM# single number following 01 page 39 / total 90 pages 1013264167-0 201242597 It will be apparent to those skilled in the art that the compound of formula VIIIA becomes the corresponding b-diketone. The rearrangement of the compound of formula IXA is known as Baker-Wen Baker-Venkataraman rearrangement (J. Chem. Soc., 1381 (1933) and Curr. Sci., 4, 214 (1933)). The base used in the process step (c) may be selected from the group consisting of: a hexamethyldidecylamino chain, a sodium hexyl decylamino group, a potassium hexamethyldidecylamino group, sodium hydride, and potassium hydride. The preferred base is a hexamethyldidecylamino lithium ruthenium. The dealkylation agent used in the step (e) for the dealkylation of the compound of the formula IXA may be selected from the group consisting of pyridine hydrochloride, boron tribromide, and trifluorobenzene. Boron B puzzle and three gasified aluminum. A preferred dealkylating agent is pyridine hydrochloride. The preparation of the starting compound of formula VIA involves the reaction of methyl-4-piperidine with a solution of 1,3,5-trimethoxy in glacial acetic acid to yield the diterpene-4-(2, 4, 6- Trimethyloxy)-[[2,3,6-tetrahydroacridine, which reacts with difluorinated ruthenium, coded sodium hydride, and tetrahydroanthracene to yield 1-methyl- 4-(2,4,6-trimethoxyphenyl) brigade _3-alcohol. 1__Methyl 4 (2,4,6-dimethylmercapto) base _3_alcohol becomes a molecular formula γι' The conversion of a compound involves an oxygen nucleophile (such as triethylamine, pyridine, potassium carbonate or In the presence of sodium sulphate, in the presence of an oxygen nucleophile, in the presence of an oxygen nucleophile, in the presence of an appropriate reagent (eg, sulfonium sulfonium, decane sulfonium, trifluorosulfonate or pentoxide) For example, sodium acetate or potassium acetate in an alcohol solvent (for example, isopropanol, ethanol or propanol) will be present in the compound diterpenoid-4-(2 4 6-trimethoxy) group by condensing ring action. The m-ol (iv) (iv) is converted to a leaving group such as a methyl group, a methanesulfonyl group, a triflate or a halide. 1011〇84#单单A0101 Page 4/90 pages 1013264167-0 201242597 β) ( + )-trans-2-(2-phenylphenyl)-5,7-di-carbyl-8-(2 - Preparation of hydrazino-1-methyl-pyrrolidin-3-yl)- benzopyran-4-one hydrochloride (Compound A) to melt the alpha ratio bite hydrochloride (4.1 g, 35.6 Ment) to (+)-trans-2-(2-carbo-phenyl)-8-(2-pyridylmethyl-1-methyl-α than succinyl-3-yl)-5,7 -Dimethoxy-stupidin-4-one (〇.4 g, 0.9 mmol) in 'and heated at 180 ° C for 1.5 h. The reaction mixture was cooled to 25 <0>C, diluted with MeOH (10 mL) and basified to pH 10 using Na. The mixture was filtered' and the organic layer was concentrated. The residue was suspended in water (5 mL) and stirred for 30 min, filtered and dried to give compound (+)-trans-2-(2-carbo-phenyl)-5,7-dihydroxy-8 -(2-Light-methylmethyl-1-indenyl-π-Bista-3-yl)-benzoin-4-yl. Yield: 0. 25 g (70%); IR (KBr): 3422, 3135, 1664, 1623, 1559 cra-1; 1H NMR (CDC13, 300MHz): δ 7.56 (d, 1H), 7.36 (m, 3H ), 6.36 (s, 1H), 6.20 (s, 1H), 4.02 (ra> 1H), 3.70 (m, 2H), 3.15 (m, 2H), 2-88 (m, 1H), 2.58 (s, 3H), 2.35 (m, 1H), 1-88 (m, 1H); MS (ES+): m/z402 (M+l); Analysis: C21H20C1NO5 C, 62.24 (62.71); H, 5.07 (4·97) ); N, 3.60 (3,48); Cl, 9.01 (8.83). The compound obtained above (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 was cooled and the solid was filtered to give the compound (+)-trans-2-(2-phenylphenyl)-5,7-di-propyl-8-(2-hydroxymethyl-1-methyl - pyrrolidin-3-yl)-benzopyran 10110847^.Single number A0101 Page 41 of 90 Page 1013264167-0 201242597 -4-Hydrazine hydrochloride. Yield: 0.21 g (97%); mp: 188 - 1 92 °C; [a]D25 = +21.3° (c = 0.2, methanol); 1H NMR (CD30D, 300MHz): δ 7.80 (d, 1H) ), 7.60 (in, 3H), 6.53 (s, 1H), 6.37 (s, 1H), 4.23 (m, 1H), 3.89 (m, 2H), 3.63 (m, 1H), 3.59 (dd, 1H) , 3.38 (in, 1H), 2.90 (s, 3H), 2.45 (m, 1H), 2.35 (in, 1H); MS (ES + ): m/z 402 (M +1) (free base). This compound was subjected to palm HPLC. Palm chromatography was performed using a column column chiral column OD-H (250 X 4. 6 ram) and a solvent system hexane:ethanol (92:08) with TFA (0.4%). The results were recorded at 264 nm at a solvent flow rate of 1 mL/min. As depicted in Figure 3, the palm HPLC showed 100% e.e. of compound (+)-trans-2-(2- gas-phenyl)-5,7-di-yl-8- (2-Pentyl-methyl-1-indolyl-pyrrolidin-3-yl)-benzopipene-4-one hydrochloride. C) ( + )-trans- 2-(2- gas-4-trifluoromethyl-phenyl)-5,7-di-butyl-8-(2-hydroxymethyl-1-indenyl-pyrrole Preparation of pyridine-3-yl)-benzopiperan-4-one hydrochloride (Compound B) The compound (+)-trans-2-(2- gas-4-trifluoromethyl)- 8-(2-Hydroxymercapto-1-indolylpyrrolidin-3-yl)-5,7-dimethoxy-benzopyrazine-4-(0.25 g, 0.5 mmol), ° ratio biting salt A mixture of the acid salt (0.25 g, 2.16 mmol) and a catalytic amount of quinoline was heated at 180 ° C for a period of 2 · 5 hours. The reaction mixture was diluted with MeOH (25 mL) and basified to pH 10 with solid Na. The reaction mixture was filtered and rinsed with methanol. The organic layer was concentrated, and 0.1% ammonia was used in the gas imitation. The m_the production number page 42/90 pages 1013264167-0 201242597 and the 4.5% methanol were used as the eluent, and the column was used. The residue was purified by chromatography to give the compound (+)-trans-2-(2-carb-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2- Hydroxy-methyl-1-indolylpyrrole bite _3-yl)-benzohethane _4_嗣. Yield: 0.15 g (63.7%); 1H NMR (CDC13, 300MHz): <5 7.99 (m, 2H), 7. 83 (d, 1H), 6. 65 (s, 1H), 6 41 (s, 1H), 4.24 (m, 1H), 3.90 (m, 2H), 3.70 (m, 1H), 3.60 (in, 1H), 3.41 (ra, 1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.28 (m, 1H); MS (ES+): m/z 470 (M+l). The compound obtained above (0.1 g, 0.2 mmol) was suspended in methanol (2 mL) and treated with etherified HCl and organic solvent evaporated to yield compound (+)-trans-2-(2- -4-trifluoroindolyl-styl)-5,7-di-transyl-8-(2-transmethylmethyl-1-methyl-π ratio _3_yl)_ benzo-bond- 4-keto hydrochloride. Yield: 0. lg (92. 8%); Ο 1H NMR (CDC13, 300MHz): 5 8.02 (d, 2H), 7.83 (d, 1H), 6.64 (s, 1H), 6.41 (s, 1H), 4.23 (ra, 1H), 3.73 (m, 2H), 3.68 (m, 1H), 3.51 (m, 1H), 3.39 (m, 1H), 2.99 (s, 3H), 2.54 (m, 1H), 2.31 (m, 1H). In vitro study involving the use of a dual combination consisting of Compound A and erlotinib Example 2: 10110847# Single number using propidium iodide (PI) in cytotoxicity assay for Compound A and Ero A0101 Page 43 / Total 90 pages 1013264167-0 201242597 Combination study of fentanyl propidium iodide fluorimetry (PI) was performed according to the procedure mentioned in Anticancer Drugs, 1995, 6, 522-32, which is incorporated herein by reference. . This assay was developed to determine the in vitro growth characteristics of human tumor cell lines and to test the cytotoxic activity of anticancer agents ("test compounds"). Propidium iodide (PI) is used as a dye that penetrates only the damaged cell membrane. The embedded complex is formed by PI and double-stranded DNA, which causes amplification of the fluorescence. After the cells were frozen at -70 °C for 24 h, PI had entered total DNA leading to the total number of cell populations. Background readings were obtained from cell-free wells containing only medium and propidium iodide. Human pancreatic cancer cell lines, Panc-Ι and AsPc-1 (derived from ATCC (American Type Culture Collection)) were seeded at a density of 1 500-3000 cells/well in 180 pL of MEM in 96-well plates (minimum Essential medium 'SAFC, USA' and RPMI 1460 (Sigma, USA) with 10% FCS (fetal calf serum) and cultured for approximately 16 h to allow cell attachment. As shown in Figures 1 a, 1 b, 2a and 2b, then different concentrations of Compound A corresponding to IC3Q, IC5Q or IC7() and erlotinib corresponding to different concentrations of IC30, IC50 or 1C70 ( LC Laboratories, USA) Treat the cells either at different concentrations (IC3〇, IC5〇 or 1C7〇) of Compound 4 or separately at different concentrations (IC3〇, 1(:5〇 or 1(:7〇) angstroms The cells were treated with rotinib. Compound A for Panc-Ι and AsPc-1 cells and ic 30, IC5() and IC7〇 concentrations of erlotinib are specifically described in Table 3 as presented herein below. The treatment period is 7 2 hours or 9 6 hours. The culture tray is cultured at 37 ° C ± 1 ° C, humidified 5% (: 〇 2 incubator. To transport the tool (DMS0 10110847#单号A0101第44页/ Total 90 pages 1013264167-0 201242597 ) Process control group holes. At the end of the culture period, remove the medium from the hole and rinse with saline buffered saline (PBS). Add 100 pL of PI working solution per well ( 7 pg/mL), and store the plate at -80 ° C for about 16 hours. Dissolve the plate and use The POLARstar optima plate reader (USA) measures fluorescence at 536 nm excitation and 590 nm divergence. Calculate the percent inhibition (%) and use the Compusyn software (

ComboSyn, Inc. USA) determines the combination index. see

Therapeutical Basis, Experimental Design f) and Computerized Simulation of Synergism and

Antagonism in Drug Combination Studies, CHOU, Ting-Chao. Pharmacol. Rev., vol. 58, no. 3, pgs. 621-681 (2006), which is incorporated herein by reference. The combination index (C·I) is used to assess the synergy between two or more compounds. C. I &lt; 1 represents a synergistic effect of the combination, c. j =1 represents that the combination is additive, and C I represents that the combination is antagonistic. In a specific embodiment of the invention, c. j is <1. (Prepare 丨 乩 乩 pi reserve solution by dissolving 1 mg of PI in ! mL of distilled water. Prepare the working solution by adding 140 of this stock solution to pBS and making up the volume to 220 mL. (7 call / 眈)) The results are presented in the following tables la, lb, 2a and 2b. In addition, the graphs in the first and the lb diagrams provide 12 and 11 graphs of single and combined doses of Compound and Erlotinib in the Panc-"cells at the end of 72 hours and (10) hours, respectively. Figure 10110847# Single No. A01〇l Page 45 of 9 for the single- and combined doses of Compound A and erlotinib in AsPc-1 cells at 72 hours and 96 hours, respectively. See page 1013264167-0 201242597. In the following tables la, lb, 2a and 2b, the symbol "+" represents the simultaneous use of an anticancer agent (test compound). Table la - Compound A was calculated for the combination index (C. I) of the combination of Compound A (at different concentrations) with erlotinib (at different concentrations) in Pane-1 cells after 72 h of treatment and Combination index of erlotinib (CI) 値 IC30 compound A + IC5 〇 erlotinib 0.5 IC30 compound A + IC7Q erlotinib 0.2 ic50 compound A + rc30 erlotinib 0.8 IC50 compound A + IC5G erro Tini 0.4 IC50 Compound A+IC7Q Erlotinib 0.1 IC70 Compound A+IC3G Erlotinib 0.7 IC70 Compound A+IC5G Erlotinib 0.5 ic70 Compound a+ic7C Erlotinib 0.2

Table lb - Calculation of the combination index (C·I) for the combination of Compound A (at different concentrations) with erlotinib (at different concentrations) in Pane-Ι cells after 96 h of treatment Compound A and erlotinib combination combination index (CI) 値 IC30 compound eight +1 (: 50 erlotinib 0.5 IC30 compound eight + 1. 70 erlotinib 0.1 IC50 compound A + IC3G erlotinib 0.8 IC50 Compound A+IC5Q Erlotinib 0.4 IC50 Compound A+IC70 Erlotinib 0.1 IC70 Compound A+IC3G Erlotinib 0.7 IC70 Compound A+IC5Q Erlotinib 0.4 ic70 Compound A+IC7G Erlotinib 0.1 Page 46/90 pages 1013264167-0 Xie Brain #单编号除1 201242597 [0012] Table 2a - After 72 h of treatment, Compound A (at different concentrations) and erlotin in AsPc-1 cells Combination of Ni (in different concentrations) • Combination index (C. I) Calculate the combination index (CI) of compound A and erlotinib 値 IC30 compound A + IC3G erlotinib 0.6 IC30 compound a + ic50 angstrom Rotinib 0.3 IC50 Compound A+IC30 Erlotinib 0.3 IC50 Compound A+IC5G Erlotinib 0.3 IC70 Compound A + IC3G erlotinib 0.5 IC70 Nigeria a + ic50 compound erlotinib 0.6

Table 2b - Calculation of the combination index (C. I) for the combination of Compound A (at different concentrations) with erlotinib (at different concentrations) in AsPc-1 cells after 96 h of treatment Combination Combination Index (CI) 値 IC30 Compound a+ic30 Erlotinib 0.4 IC30 Compound A+IC5Q Erlotinib 0.1 IC50 Compound A+IC3Q Erlotinib 0.3 IC50 Compound A+ IC5G erlotinib 0.2 IC70 compound A+IC30 erlotinib 0.4 IC70 compound A+IC50 erlotinib 0.4 [0014] Table 3 - Compound A and erlotin in Panc-Ι and AsPc-1 cell lines 30, 50, and 70 percent inhibition concentrations (ICQn, ICKn, and

0 UDU 10110847#单号A〇101 Page 47/Total 90 Page 1013264167-0 201242597 Cell strains Wf# r ..τΰΓ\——--———— wia. v. μΜ ) ------ —j 儿3〇1^50 IC70 Panc-1 A 0.4 0.6 0.7 埃罗#尼'圆————— 5 12 22 — AsPc-1 Compound A — 0.7 1.5 3.0 Erlotinib 10 ——— 30 - In-vitro study involving the use of a triple combination of Compound A, erlotinib/lapatinib, and gemcitabine Example 3: Materials: Gemcitabine, Lapatinib, and Erlotinib were obtained from LC Laboratories, USA . The CCK-8 cytotoxic kit was manufactured by D〇 jind〇 Molecular Technologies, Japan. Medium and fetal bovine serum (FBSpha &amp; Sigma (Si:.Louis, M〇h:^Gibco (Paisley 'Scotland)). Panc — i and Mia Paca 2 cells were obtained from the American Fe Conservation Center (ATCC, Manassas, VA). The cells were maintained in a sterile, penicillin-streptomycin solution, sterile filtered, with 5, oxime units of penicillin and 5 mg streptomycin/mL (P-4458, Sigma-Aldrich, Dulbecco's Modified Eagle Medium (MEM) in the US. The cells were grown in 75-cm2 flasks and maintained in a humidified (37 ° C, 5% CO 2 ) incubator. 80% of the fullness. Cell proliferation assay: The logarithmically grown cells were plated at a density of 3 X 103 cells/well and allowed to recover overnight. Different concentrations of different anticancers ranging from 10 nM to 30 μΜ The stimulator (Gemcitabine, Compound A, lapatinib, and erlotinib) stimulating the cells, and the control group cells receiving a single number A0101 page 48 / a total of 90 pages 1013264167-0 201242597 having 10% serum, containing 0.2% Concentration of dimethyl sulfoxide (DMSO) transport Dulbecco's modi ed Eagle's medium (DMEM). After 72 hours, cytotoxicity was determined by cck-8 reagent (Dojindo Molecular Technologies, Japan); (WST-1 [2-(2-methoxy-4-phenyl succinylbenzene) ))-3-(4-Butylphenyl)-5-(2,4-diphenyl)]-2H-tetrazolium monosodium salt analysis method. Add 5μ1/ according to the manufacturer's instructions The wells were CCK-8 reagent and the plates were incubated for 2 hours. The absorbance was measured at a wavelength of 450 nm corrected to 650 nm on a Tecan Sapphire multiplexed microplate reader (Tecan, Germany, GmbH). Toxicity was determined and normalized to the control group. CCK-8 (Cell Count Kit-8) non-radioactive colorimetric assay was performed to determine the in vitro growth characteristics of Panel and Mia Paca2 cells, and to test anticancer agents, gemcitabine, The cytotoxic activity of Compound A, lapatinib and erlotinib when used in combination ^ CCK-8 allows a convenient analytical method using Dojindo's tetrazolium salt, WST 8-(2-(2-曱oxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt Which generates a water-soluble formazan dye Juan after biological reduction in the presence of an electron carrier (1-methoxy PMS) is. The CCK-8 solution was added directly to the cells; no premixing of the ingredients was required. CCK-8 is a sensitive non-radioactive colorimetric assay and cytotoxicity assay for determining the number of viable cells in cell proliferation. WST-8 is reduced by the cell dehydrogenase bios to an orange quinone product which is soluble in the tissue culture medium. The amount of sputum produced is directly proportional to the number of viable cells. The detection sensitivity of the cell proliferation assay using WST-8 is higher than that using other tetrazolium salts, such as MTT, XTT, MTS or WST-1 (PCT Patent Publication No. 1013264167-0 Page 49 of 90 Page 201242597 opened W097/38985). The optical density was determined at a measurement wavelength of 450 nm and a reference wavelength of 630 nm.

Determination of 50% inhibitory concentration (IC^n) of Compound A, erlotinib/lapatinib and gemcitabine in Panc-Ι cells, Mia Paca-2 cells, HPAC cells and Capanl cells

DU To determine the specific anticancer agents of the following concentrations of gemcitabine, compound A, erlotinib, and lapatinib in ic5Q' in Panc-Ι cells, Mia Paca-2 cells, HPAC cells, and Capan1 cells (" The test compound") treats the cells. All anticancer agents at the following concentrations of 〇.03, 0.1 μΜ, 0.3 μΜ, 1 μΜ, 3 μΜ, 10 μΜ, 30 μΜ, and 100 μΜ were analyzed for their ability to exhibit cytotoxicity, particularly 0% cytotoxicity. The cells were seeded at a density of 3 〇〇〇 cells/well in a 96-well plate at a tissue culture level of 200 μg, and the cells were placed in a humidified 5% ± 0.2 cm incubator at 37 吣 ± 0.50 ( : lower recovery L· complex 2 4 hours. 2 4 hours later '1 μL of 200 Χ test compound (gemcitabine' compound a, erlotinib and lapatinib dissolved in pure dimercapto (IA) The concentration of the final DMSO in the well is 0.5%. The culture plate is placed in a humidified 5% ± 0.2 C02 incubator at 37 ± Incubate for 48 hours at 5 ° C. After 48 hours, remove the plate from the C 2 incubator and add 5 pL of cell count kit (CCK-8) to each well. Maintain the same plate. At 370C for 3 hours and let it return to room temperature "Read the absorbance at 450 nm wavelength on Tecan Multimode safire. Calculate the percent cytotoxicity using the equation below. Percent cytotoxicity = @MS〇 control group OD - treatment fine ΤΩΤη X100)

DMSO Controlled OD 10110847# Single Number i A0101 Page 50 / Total 90 Page 1013264167-0 201242597 [0015] Dose-response studies at 72 hr in panc-1 cells showed gemcitabine, Compound A, erlotinib And lapatinib inhibited 50% growth (IC5()) at 0. 0 6 μ Μ, 0.51 μΜ, 7. 0 μΜ, and 4.8 μμ, respectively. The results are presented in Table 4a and plotted in Figure 3a. Dose-response studies at 72 hr in MiaPaca-2 cells showed inhibition of gemcitabine, Compound A, erlotinib, and lapatinib at 0.22 μΜ, 0.72 μΜ, 9.0 μΜ, and 7.6 μΜ, respectively. % growth (IC5()). The results are presented in Table 4b and plotted in Figure 3b. Dose response studies at 72 hr in HPAC cells showed that gemcitabine, Compound A, and erlotinib inhibited 50% growth (IC5Q) at 1.6 μΜ, 1.1 μΜ, and 2. 3 μΜ, respectively. The results are presented in Table 4c. u Dose response studies at 72 hr in Capanl cells showed that gemcitabine, Compound A, and erlotinib inhibited 50% growth (IC5Q) at 2.9 μΜ, 3.6 μΜ, and 3.9 μΜ, respectively. The results are presented in Table 4d. Similarly, ic30, ic7Q, and ic90 concentrations were established for all test compounds (anticancer compounds) from doses in which specific compounds showed 30%, 70%, and 90% activity in cytotoxicity assays, respectively. Table 4a - 30%, 50%, 70%, and 90% inhibitory concentrations of gemcitabine, Compound A, erlotinib, and lapatinib in Panc-1 cells (IC30, IC50, %〇, and I,) [0016 Anticancer agent Panc-1 cells (inhibitory concentration μΜ) IC3〇IC5〇IC7〇IC90 complex A 0.1 0.5 3.1 9.4 Gemcitabine 0.02 0.06 3 7.3 Erlotinib 1.6 4.8 12 26 Lapatinib 2.8 7 10 35 51 Page / Total 90 pages 1084#单单A_ 1013264167-0 201242597 [0017] Table 4b - 30%, 50%, 70% of gemcitabine, Compound A, erlotinib and lapatinib in MiaPaca-2 cells and 90% inhibitory concentration (IC3〇, ^5〇, IC7〇, and ICg〇) Anticancer agent MiaPaca-2 cells (inhibition of 翻μ?) ~ ---~~--- IC3〇IC5O IC7〇' IC9〇 Compound A 0.3 0.72 5.3 ---- 9.5 Gemcitabine 0.05 0.22 3.1 ----~~ 7.1 Erlotinib 2.6 7.6 15 &quot;----- 34 Lapatinib 3 9 20 ~~---- 45 [0019 Table 4c - 30%, 50%, 70%, and 90% inhibitory concentrations of gemcitabine, Compound A, erlotinib, and lapatinib in HPAC cells (ic 30, IC5〇, iC7〇, and 1C90) HPAC cells Inhibitory concentration μΜγ-- 1.30 IC5〇IC7〇IC90 complex A 0.4 1.1 —-- 4.6 21.4 Gemcitabine 0.9 1.6 8.7 28.9 Erlotinib 1.4 2.3 6.3 10.7 [0021] Table 4d - Gemcitabine, Compound A in Capanl cells 30%, 50%, 70%, and 90% inhibitory concentrations of erlotinib and lapatinib (IC30 'IC50, 1. 7〇, and ICg〇) Anticancer agent Capanl * (inhibitory concentration μΜ) —~ ' Ic3〇IC5〇IC70 IC9O conjugate A 1.6 3.6 7.8 22.3 Gemcitabine 1.1 2.9 11.7 23.7 Erlotinib 1.1 3.9 12.6 20.6 Page 52 of 90 [0022] 1013264167-0 10110847 产单编# A0101 201242597 [0023] Example 4: Combination of gemcitabine with Compound A and erlotinib or with Compound A and Lapatinib in Panc-1 cells for a final concentration of 0·02 μΜ ( IC3〇) or 〇. 〇6 μΜ ( 1C ) Dosage of gemcitabine, 0.1 μM (IC3Q) final concentration of Compound A, 2.8 μΜ (IC3()), final concentration of lapatinib, and 1.6 μΜ (IC3G) final concentration of erlotinib A continuous combination of the three specific anticancer agents. Subsequent processing sequences were as follows: Panc-1 cells were treated with gemcitabine at IC or 30 IC5() for up to 24 hours. At the end of 24 hours, the cells were washed twice in non-doped minimal essential medium (MEM). Fresh minimal essential medium with 10% serum (200 μίν well) was added, followed by treatment with compound guanidine and erlotinib or Compound A and lapatinib from 24 hours to 96 hours. Cytotoxicity was measured using the CCK-8 method at 96 hours. Use the Combosyn software to determine the combination index. The combination index (C.I) is used to assess the synergy between two or more compounds. CI &lt;1 represents a synergistic effect of the combination, CI = 1 represents that the combination is additive, and CI &gt; 1 represents that the combination is antagonistic. Gemcitabine at IC3Q concentrations was found to be synergistic with Compound A and lapatinib at a combination of 1 (30 concentrations) per compound. Gemcitabine at 1〇30 showed 12% cytotoxicity, and at 1C The combination of Compound A and lapatinib IC3Q showed 26% cytotoxicity. However, it was noted that when treated with gemcitabine ICqn for 24 hrs, followed by 1 (: „ 〇 〇 U 3 A complex with IC3 () The combination of lapatinib for 72 hours of combined use showed a 73% increase in cytotoxicity, which was 35% more cytotoxic than the additive effect, suggesting that the three antibodies were in Panc-1 cells. Synergism between cancer agents. The results are presented in Table 5a and plotted in Section 4a 1013264167-0 10110847#单号A01〇l Page 53 of 90 201242597. Discovered gemcitabine with Compound A and erlot The combination of nisin has a synergistic effect at the ic3 〇 concentration of each anticancer agent. At 1 (: 3 〇 gemcitabine shows 12% cytotoxicity, and the compound Α 与 c and erlotinib J c 3 〇 , 30 combination shows 28% cytotoxicity However, when treated with i c concentration of gemcitabine for 24 hrs, followed by a combination of ic3 () concentration of compound human and erbium (9) concentration of erlotinib for 72 hours, showed an increase in cytotoxicity of 72%. It is 32% more cytotoxic than the additive effect, suggesting a synergy between the three anticancer agents in Panc-Ι cells. The results are presented in Table 5a and plotted in Figure 4a. In the middle, the symbol "+" represents the simultaneous use of an anticancer agent (test compound). Table 5a - IC3{) concentration of gemcitabine with compound a and erlotinib or with compound A and lapatinib in Panc-Ι cells Combination Study No. Anticancer Agent % Cytotoxic Combination Index (CI) 値1 Gemcitabine IC30 12 2 Compound aic30 13 - 3 Lapatinib IC30 14 - 4 Compound A ic30 + Lapatinib IC30 26 - 5 Erlotinib IC30 15 - 6 Compound A rc30 + Erlotinib IC30 28 - 7 Gemcitabine ic30 + (Compound A IC30 + Lapatinib IC30) 73 0.44 8 Gemcitabine IC3C+ (Compound A ic3() + Erlotinib IC30) 72 0.17 3 0 [0024 ]

The combination of gemcitabine at IC5Q concentration with compound A at IC3() concentration and lapatinib at 1C concentration was found to have a synergistic effect in Panc-1 cells. IC50 concentration of gemcitabine showed 21% cytotoxicity, as well as ic 30 10U084# single number A0101 page 54 / total 90 pages 1013264167-0 201242597 concentration of compound A and IC3 () concentration of lapatinib combination showed 26 % cytotoxicity. However, when treated with I(^n concentration of gemcitabine for 24 hrs) followed by a combination of IC3Q concentration of Compound A and IC3() concentration of lapatinib for 72 hours, it showed an increase in cytotoxicity of 89%. 'It has 42% more cytotoxicity than the additive effect, suggesting a synergistic effect between the three anticancer agents in Panc-Ι cells. The results are presented in Table 5b and plotted in Figure 4b. 1C concentration of gemcitabine with 1C concentration of Compound A and 1C. The combination of erlotinib in concentration has a more synergistic effect in Pane-1 cells. IC5() concentration of gemcitabine shows 21% cytotoxicity, and IC30 concentration The combination of Compound A and IC3() at a concentration of erlotinib showed 28% cytotoxicity. However, when treated with ic concentration of gemcitabine

0 U 24 hrs, followed by a combination of IC3Q concentration of Compound A and IC3Q concentration of erlotinib for 72 hours, showed an increase in cytotoxicity of up to 86%, which was 37% more than the additive effect. Cytotoxicity, suggesting a synergy between the three anticancer agents in Pane-Ι cells. The Q results are presented in Table 5b and plotted in Figure 4b. Table 5b - Combination of gemcitabine with compound a and erlotinib or with compound A and lapatinib in Pane-Ι cells 1〇ll〇847#单单号A0101 Page 55 of 90 1013264167-0 201242597 [0025] No. Anticancer agent % cytotoxicity ^ Combination index __(ci) 値 - _ 1 Gemcitabine IC50 21 1 Complex Arc30 ' — ~~~~~~: —__ 3 Lapatinib iC3 〇14 -——_ 4 also a compound A IC30 + lapatinib IC30 —— 26 ~^--- 5 erlotinib ic30 ^ 6 compound AIC30 + erlotinib IC30 ---------- - _ 28 一——S ---__ 7 Gemcitabine IC5G+ (Compound A IC3〇 + Lapatinib IC30) 89 ~~~~- 0.19 8 Gemcitabine IC50+ (Compound A IC30 + Erlotinib IC30) ~~~^ ~~~-0.11 Example 5: Combination of gemcitabine with compound hexazone and erlotinib or with compound A and lapatinib in MiaPaca-2 cells for a final concentration of 0.05 μΜ (IC3〇) and 0.22 μΜ ο,) The dose of gemcitabine, 0.3 μΜ (Ι(:3.) final concentration of the compound concentration, 3·0 μΜ (Ι〇3{)), the final concentration of lapatinib, 2 6 υΜ (IC) The final concentration of erlotinib was analyzed for the sequential combination of the three specific anticancer agents described above. The subsequent treatment sequence was as follows: MiaPaca2 cells were treated with IC or 丨30-50 gemcitabine for 0 to 24 hours. At 24 hours, sisters 'The cells were washed twice with a non-doped minimum essential medium (Mem). Fresh minimal essential medium with 10% serum (2 〇〇 UL/well) was added, followed by Compound A from 24 hours to 96 hours. Treatment with erlotinib or Compound A and lapatinib. At 96 hours, the cytotoxicity was measured using the cck-8 method. The combination index was determined using the Combosyn software. The combination index (C · I ) was used to evaluate 2 or more. Synergism between the compounds. CI &lt;1 means that the combination has a synergistic effect, CI = 1 means that the combination is additive' and CI &gt; 1 means that the combination is antagonistic. Page 56 of 90 1013264167- 0 201242597

The combination of gemcitabine at IC3() concentration with Compound A and Lapatinib at the ic30 concentration of each compound was found to be synergistic. Gemcitabine at 1C concentration showed 16% cytotoxicity, as well as at 1C

Q U concentration of compound A with yttrium (yt concentration of lapatinib (the combination of η concentration y U 3 0 showed 26% cytotoxicity. However, when treated with 1C of gemcitabine)

〇U 24hrs, followed by a combination of IC3() compound A and lapatinib in IC3() for 72 hours, showed an increase in cytotoxicity of 64%, which is more than the addition effect. % cytotoxicity, suggesting a synergy between the three anticancer agents in MiaPaca-2 cells. The results are presented in Table 6a and plotted in Figure 5a. The combination of gemcitabine at IC3Q concentration with Compound A and erlotinib was found to have a synergistic effect at the ICqn concentration of each compound. The concentration of gemcitabine showed 16% cytotoxicity, and the combination of Compound A at ICQn concentration and erlotinib at IC3() concentration showed 27% cytotoxicity. However, when treated with gemcitabine at an IC3() concentration for 24 hrs, followed by a combination of IC3 () concentration of Compound A and IC3() at a concentration of erlotinib for 72 hours, it showed a cytotoxicity of 69%. Increased, it is 26% more cytotoxic than the additive effect, suggesting a synergistic effect between the three anticancer agents in MiaPaca-2 cells. The results are presented in Table 6a and plotted in Figure 5a. In the table below, the symbol "+" represents the simultaneous use of an anticancer agent (test compound). Table 6a - Combination of gemcitabine with compound A and erlotinib or with compound A and lapatinib in MiaPaca-2 cells 101 face 7 dream number A () 101 page 57 / total 90 pages 1013264167-0 201242597 No. Anticancer agent % cytotoxicity combination index (CI) 値 1 Gemcitabine 丨 c30 16 . 2 Compound A IC30 14 3 Lapatinib IC30 17 4 Compound A K30 + Lapatinib IC30 26 5 Ero尼ini 1C30 19 6 Compound AIC30+ Erlotinib IC30 27 7 Gemcitabine K:30+ (Compound A ICBoigiftf nyic ic30) 64 0.29 8 Gemcitabine IC30+ (Compound A IC30 + Erlotinib K: 3〇) 69 0.32 Found IC5〇 The combination of gemcitabine at a concentration of Compound A at a concentration of 1c3 and lapatinib at an IC3() concentration is more synergistic in MiaPaca2 cells. The IC5Q concentration of gemcitabine showed 21% cytotoxicity, and the combination of IC3 oxime concentration of Compound A and IC3Q concentration of lapatinib showed 26% cytotoxicity. However, when treated with gemcitabine at an IC5Q concentration for 24 hrs, followed by a combination of IC3 (concentration of Compound A and IC3() at a concentration of lapatinib for 72 hours, an increase in cytotoxicity of 91% was observed, It is 44% more cytotoxic than the additive effect, suggesting a synergistic effect between the three anticancer agents in MiaPaca-2 cells. The results are presented in Table 6b and plotted in Figure 5b. The combination of gemcitabine (IC5Q) with Compound A (IC3Q) and erlotinib (IC3()) was found to be more synergistic in MiaPaca2 cells. Gemcitabine at a concentration of 1匚5〇 showed 21% cytotoxicity, and a combination of 1 (^. concentration of Compound A with IC3() at a concentration of erlotinib showed a cytotoxicity of 27.7%. However, when 1C The concentration of gemcitabine was treated for 24 hrs, followed by a combination of IC3 () concentration of Compound A and IC3 () concentration of erlotinib for 72 hours, showing an increase in cytotoxicity of 88%. The effect is 40% more cytotoxic, suggesting that in 1013264167-0 1Q11Q847f single number A0101 page 58 / total 90 pages 201242597

Synergism between the three anticancer agents in MiaPaca-2 cells. The results are presented in Table 6b and plotted in Figure 5b. In the table below, the symbol "+" represents the simultaneous use of an anticancer agent (test compound). Table 6b - Gemcitabine and Compounds at ICEn Concentration in MiaPaca-2 Cells

DUA and erlotinib or combination with compound A and lapatinib number anticancer agent % cytotoxicity combination index (CI) 値 1 gemcitabine IC50 21 - 2 compound AIC30 14 - 3 lapatinib ic30 17 - 4 Compound aic3〇+lapatinib IC30 26 - 5 Erlotinib ic3〇19 - 6 Compound AIC30+ Erlotinib IC30 27 - 7 Gem IC5Q+ (Compound A IC3Q + Lapati Ik IC^n ) 91 0.14 8 Gem IC5 〇+ (Compound A IC3G+Errotin fp, IC-ϊπ) 88 0.15

[0027] Example 6: Combination study of gemcitabine with Compound A and erlotinib in HPAC cells found that the combination of gemcitabine with Compound A and erlotinib synergistically at the ic3() concentration of each anticancer agent, respectively. effect. The ic3 () concentration of gemcitabine showed 17% cytotoxicity, and the combination of compound A at IC3() concentration and erlotinib at IC3() concentration showed 33% cytotoxicity. However, when treated with an IC3() concentration of gemcitabine for 24 hrs, followed by a combination of IC3() concentration of Compound A and IC3() concentration of erlotinib for 72 hours, it showed 88% cytotoxicity. Increased, it is 38% more cytotoxic than the additive effect, suggesting a synergistic effect of a combination index (C.I) of 0.21 between the three anticancer agents in HPAC cells. The results are presented in Table 7 and plotted in Figure 6. Table 7 - Gemcitabine and Compounds at ICgn4ICKn Concentration in HPAC Cells

〇U 〇U 10110847#单单皿01 Page 59/Total 90 Page 1013264167-0 201242597 A combination of A (in IC3Q or IC5Q concentration) and erlotinib (in IC3() or IC5() concentration) Anticancer agent at 96 hr cytotoxicity % combination index (CI) 値 day 1 day 2 1 gemcitabine IC30 - 17.29 - 2 gemcitabine IC50 - 21.82 - 3 compound A IC30 - 16.95 - 4 compound aic50 - 23.48 - 5 erro尼尼ic30 - 14.21 - 6 erlotinib IC50 - 24.84 - 7 gemcitabine IC30 complex A IC30 51.83 0.92 8 gemcitabine IC30 compound aic50 62.37 0.57 9 gemcitabine IC50 compound a IC30 63.47 0.66 10 gemcitabine IC50 compound aic50 71.47 0.42 11 erlot尼30 Compound A: IC30 47.44 0.96 12 Erlotinib κ30 Compound aic50 54.99 0.65 η Erlotinib IC50 Compound ακ:30 64.10 0.88 14 Erlotinib IC50 Compound aic50 73.37 0.59 15 Gemcitabine Compound a IC3〇+Ero Ίίκ IC-^n 33.29 - 16 Gemcitabine Compound A IC3G+Erodide^ IC.n 39.94 _ 17 Gemcitabine IC30 Compound A IC3〇+Erodinib IC30 88.42 0.21 18 Gemcitabine IC30 Compound A IC3〇+Erodinib 1.50 89.53 0.27 19 Gemcitabine IC50 Compound A IC3()+ Erlotinib iC30 87.34 0.57 20 Gemcitabine IC50 Compound A IC3Q+ Erlotinib Iso 96.51 0.63 [0028] Example 7 A combination study of gemcitabine with Compound A and erlotinib in Capanl cells found that the combination of gemcitabine with Compound A and erlotinib synergistically at the ic3() concentration of each anticancer agent, respectively. Gemcitabine at an ic3() concentration showed 11% cytotoxicity, and a combination of Compound A at IC3() concentration and erlotinib at IC3() concentration showed 28% cytotoxicity. However, it was noted that treatment with gemcitabine at an ICqn concentration for 24 hrs followed by a combination of Compound A at ICQi1 concentration and erlotinib at an ICgn concentration

〇U 〇U 10110847# Single No. A〇101 Page 60/Total 90 Page 1013264167-0 201242597 When combined for 72 hours, it shows a 78% increase in cytotoxicity, which is 40% more cells than the addition effect. Toxicity, suggesting a synergistic effect of a combination index (C.I) of 0.34 between the three anticancer agents in Capanl cells. The results are presented in Table 8 and plotted in Figure 7. Table 8 - Gemcitabine and compound oxime (in ICqn or Ι (: ςη concentration) and erlotinib (in ICqn4) in Capanl cells at 1 (^ or 1 (^.

ο I) a U 〇U IC5〇 concentration) combination study serial number anti-cancer agent in % hr cytotoxicity % combination index (CI) 値 day 1 day 2 1 gemcitabine IC30 - 11.78 - 2 gemcitabine IC50 - 19.67 - 3 Compound aic30 - 17.94 - 4 Compound aic50 - 28.50 - 5 Erlotinib ic30 - 17.00 - 6 Erlotinib rc50 - 27.42 - 7 Gemcitabine ic30 Compound aic30 48.00 0.81 8 Gemcitabine rc30 Compound aic50 63.81 0,9 9 Gemcitabine ic50 Compound aic30 66.01 0.47 10 Gemcitabine ic50 Compound aic50 74.07 0.71 11 Erlotinib ic30 Compound aic30 41.20 0.95 12 Erlotinib IC30 Compound aic50 47.79 1.75 13 Erlotinib IC50 Compound aic30 53.68 0.96 14 Erlotinib IC50 Compound AIC50 67.31 1.02 15 Gemcitabine-free compound A IC3〇+Erodinib 1C,η 28.14 - 16 Gemcitabine-free compound A IC3G+Erodinib IC.n 36.12 - 17 Gemcitabine IC30 Compound A IC3G+Erodinib ic,n 78.01 0.34 18 Gemcitabine IC30 Compound A IC3C) + erlotinib ic.n 84.81 0.39 19 gemcitabine IC50 Compound A IC3Q + erlotinib 1C, η 81. 66 0.45 20 Gemcitabine IC50 Compound A IC3G+ Erlotinib ΤΓ.Λ 91.06 0.46

[0029] Example 8 Determination of Protein Expression A) High Content Screening (Intratube Protein Estimation by Cellomics VTI Array Scanning) 1〇1 Brain #单编号A0101 Page 61 / Total 90 Page 1013264167-0 201242597 The density of 7.5 X i〇3 cells/well was seeded in 96-well plates. At 24 h after sowing, the minimum essential medium was replaced with fresh minimal essential medium (MEM) with 1% serum. The anticancer agent was treated at a specific concentration and the cells were cultured for 4 hours and 12 hours, respectively. At each time point, the end of the day 'to determine the protein performance' was fixed with 3.7 % furfural (Sigma St. Louis, MO) in PBS for 1 min at room temperature, followed by 0.15 % TritonX-100 (Sigma St. Louis, MO) permeabilization (permeabilization MO min. After permeabilization, cells were blocked with 5% bovine serum albumin for 2 hours. After the blocking step, human-specific primary antibody was added Up to 1 h. After incubation with the primary antibody, the nuclei were stained with Hoechst 3342 (blue) and the different proteins (PEGFRY1171 pEGFRY875 pAKT'pRB and cyclin D) were labeled by secondary anti-Zhao labeled Dylight548 (red) )

Primary antibody localization. Scan the ©¥11 HCS reader in the Cellomics array (Thermo Fisher Scientific Inc.

The plate was scanned on Waltham, MA) to determine the immunofluorescence of pEGFRY1171 pEGFRY875 pAKT, pRB and cyclin D. Cell lomics target activation and molecular translocation bio-algorithm were used to analyze all data points, and the quantitative data was expressed as a percentage (%) activation compared to the DMS0 control group. Count 1 000 cells or 20 fields of view for each of the two replicate wells and present the results as an average of ±5 £. B) Analysis of different protein expression in Panc-Ι cells using array scanning for 0. 02 μΜ (IC3Q) or 0.06 μΜ (IC5()) final concentration dose of gemcitabine, 〇. 1 μΜ (IC3() The final concentration of the compound A, 2. 8 μΜ ( IC3 () ) final concentration dose of lapatinib, 1. 6 μΜ ( 1013264167-0 10110847 ^ single _ Α () 1 ϋ 1 * 62 1 7 * 90 1 201242597 IC3〇) The final concentration of erlotinib was analyzed for a continuous combination of the three specific anticancer agents described above. Since erlotinib or lapatinib is used in the triple combination of anticancer agents, it has been called three anticancer agents. The treatment sequence was as follows; Panc-Ι cells were treated with gemcitabine of IC3() or IC5() for up to 24 hours. At the end of 24 hours, the cells were washed twice with a minimally essential medium that was not miscellaneous. Fresh minimal essential medium with 10% serum (200 μL/well) was added' followed by treatment with Compound A and erlotinib or Compound A and lapatinib for 4 hours and 12 hours, respectively. After 4 hours and 12 hours, each plate was fixed and stained for a specific protein using the same method as described above. C) Analysis of protein expression in pane-1 cells by pEGFR_Y1173 found that in combination with compound A and lapatinib or compound A with erlotinib in Pane-Ι cells in each compound (anticancer agent) Synergistic effect at the ic3Q concentration. Gemcitabine at a concentration of ic3() or ic50 and Compound A at a concentration of 1C did not show 30 inhibition of PEGFR-YU73. The single anticancer agent 'erlotinib and lapatinib at 1C concentration showed only 40% and 47% inhibition, respectively; and gemcitabine 1 (^ (with compound A and erlotinib) or ( The combination of either Compound A and lapatinib showed an inhibition of 59.2 &amp; 72.5% 6£6?1^-¥1173 in the Panc-Ι cells at 8 hours. The results are plotted in Figure 8a. D) For the protein expression analysis of pEGFR-Y845 in Panc-Ι cells, it was found that in the Panc-Ι cells, gemcitabine combined with Compound A and lapatinib or Compound A with erlotinib in each anticancer The IC3() concentration of the agent was 10110847# single, numbered fiber 01 page 63 / total 90 pages 1013264167-0 201242597 synergistic. Gemcitabine at IC3Q (iv) 5G concentration and compound a at concentration did not show inhibition of pEGFR_Y845. The single drug erlotinib and lapatinib showed only 33% and 31% inhibition, while gemcitabine IC (10) and (combination and erlotinib) or (Compound A and lapatinib) Combined in

The inhibition of the amount of 65.2 &amp; 78.2% PEGFR-Y1845 was shown in Panc-1 cells at 8 hours. The results are plotted in Figure 81). E) Analysis of the protein expression of pAKT-S473 in Panc-Ι cells found that in combination with gemcitabine and compound a and lapatinib or compound A with erlotinib in Panc-Ι cells in each anticancer agent The IC concentration of 30 has a synergistic effect. Compound A, erlotinib, and lapatinib at the concentration of IC3 () showed no significant inhibition at 12 hours, while i.e. showed a concentration of 19.5% in I[[3] and IC5〇 concentrations of gemcitabine. And inhibition of 25.7% PAKT-S473. Compound A and erlotinib and the combination of compound a and lapatinib at IC3() concentration showed only 32.9% and 29.1% inhibition' while gemcitabine IC5() and (Compound A or erlotide) The combination of either N) or [Compound A and Lapatinib] showed 62.5 &amp; 72.2%? human 1 (1'-3473 inhibition) at 12 hours in the pane- 〗 cells. Figure 8b. F) Analysis of protein expression in pnc-S780 in Panc-Ι cells. In the Panc-Ι cells, gemcitabine was combined with compound a and lapatinib or compound A with erlotinib. There is a synergistic effect at the concentration of the anticancer agent. Gemcitabine, erlotinib, and lapatinib at IC3Q and IC5 〇 concentrations did not show significant inhibition at 12 hours, whereas Compound A at 2 concentrations showed inhibition of 25% pRB-S780 at 10110847^ ^'^ A〇101 Page 64 of 90 1013264167-0 201242597. The combination of Compound A with erlotinib and Compound A and Lapatinib at IC3() concentration shows only 17% and 23.5%. Inhibition, and the combination of gemcitabine IC cn with (Compound A or Erlotinib) or (Compound A and Lapatinib) showed 卩1^- at 12 hours in Pane-1 cells, respectively. 53.4 &amp; 72.1% inhibition of the 8480 amount. The results are plotted in Figure 8d. G) Analysis of protein expression in cyclin D in Pane-1 cells. In the Pane-Ι cells, gemcitabine with Compound A and Lapa The combination of nisin or erlotinib has a synergistic effect at the Ic3() concentration of each anticancer agent. When compared to the control group, the gemcitabine at a concentration of 1 (^. or 1) and the compound A at an IC3() concentration showed a criticality of 27.7%, 20.3, and 27.8%, respectively. Inhibition. Anticancer agents, erlotinib and lapatinib alone or in combination with Compound A did not show any significant inhibition at 12 hours, while gemcitabine ICKn with (Compound A or Erlotinib) or (Compound A and The combination of either of lapatinib showed a inhibition of 69.8 &amp; 63.6% of cyclin D at 12 hours in Pane-1 cells. The results are plotted in Figure 8e. Example 9 Cleaved thiocysteine Analysis of the amount of Caspase-3 expression was performed to evaluate the mechanism by which the triple combination of gemcitabine, (erlotinib or lapatinib) and compound A blocks proliferation and whether it can induce pancreatic cancer cells. Apoptosis. Seeds were seeded in 96-well plates at a density of 7.5 X 103 cells/well. At 24 h after sowing, the minimum essential medium was replaced with fresh minimal essential medium with 10% serum. Specific as mentioned below Concentration in Pane-1 and Miapaca-2 cells Treatment of anticancer agents (gemcitabine, erlotide 1013264167-0 10110847# single number A〇101 page 65 / total 90 pages 201242597 ni or lapatinib and compound A), and cultured for 48 hours. At the end of 48 hours, To determine protein performance, cells in 96-well plates were centrifuged at 800 g for 5 minutes. The culture supernatant was removed, and 2 硫 thiocysteine-3 assay buffer was added, and the plate was again set at 800 g. Centrifuge for 5 minutes. Remove the supernatant and lyse the cells with 10 〇Crease-3 lysis buffer and shake at 300 rpm for 30 min at room temperature in a rotary shaker. Centrifuge at 8 μg for 10 minutes' and transfer 90 uL of the supernatant into a new black well plate. Add 1 μ of thiocysteine-3 receptor to the 9 μg solution. Incubate for 30 minutes at 37 C. At the end of the incubation, the plate is incubated.

The Tecan Safire multimode reader reads at 485 nm excitation wavelength and 535 nm divergence wavelength. A) The treatment mode used to evaluate the activity of thiocysteine protease-3 in Panc-1 cells for 0.02 ( IC3G) and 〇. 〇6 UM ( IC5()) final concentration agent 吉 gemcitabine, 〇. 1 μΜ (IC3()) final concentration of compound a, 2.8 μΜ ( IC3()) final concentration dose of erpentinib in rapatinib, 丨.6 (1匸3〇) final concentration agent A continuous combination of the above three anticancer agents. The treatment sequence was as follows; treatment of Panc-1 cells with 1 (:3〇 or 1 (:5〇) with gemcitabine for 0 to 24 hours. At the end of 24 hours, the cells were washed twice with non-doped MEM medium. 〇% serum (2 μμι/well) of fresh MEM, followed by treatment with Compound A and erlotinib or Compound A and lapatinib for 48 hours. After 48 hours, the thiocysteine protease was as described above. -3 Active assay for the culture plate. B) Treatment mode for assessing the activity of thiocysteine-3 in MiaPaca-2 cells 0847#单单除1 page 66/90 pages 1013264167-0 201242597 0.05 μΜ (IC3〇) and 〇.22 uM (!,) final concentration dose of gemcitabine, G.3 μΜ (IC30) final concentration dose of compound a, 3. 〇μΜ (IC30) final concentration dose of lapatinib 2. 6 μΜ ( IC ^ ) final concentration dose of erlotinib was analyzed for continuous combination of the above three anticancer agents. The treatment sequence was as follows; treatment of MiaPaCa 2 cells with gemcitabine at ^(10) or ^^ for 24 hours. At the end, rinse the cells in non-doped MEM medium. Fresh MEM with 1% serum (200 pL/well) was added, followed by treatment with compound a and erlotinib or compound A and lapatinib for 48 hours. After 48 hours, thiocysteine as described above Acidification of the culture plate by acidase-3 activity assay C) Protein expression analysis of thiocysteine-3 activity in Panc-Ι and MiaPaca-2 cells was induced by significant apoptosis, gemcitabine and Compound A The combination of lapatinib, or the combination of Compound A and erlotinib, enhances the chemo-chemical resistance of gemcitabine in pancreatic cancer cells. A single treatment with gemcitabine, erlotinib, lapatinib, and Compound A at IC3〇 or 1C5〇 did not show significant thio-Caspase-3 activity induction. When compared to the control group in Panc-Ι and MiaPaca-2 cells, Compound A was only 30% to 35% sulfur deamination with erlotinib and 30% of erpatinib at iC concentration. Acid protease 3 is activated. Gemcitabine and the compound quinone and erlotinib or lapatinib showed significant induction of up to 75% to 80% thiocysteine 3 in both cells at 48 hours. The results are plotted in Figure 9. [Simplified illustration] [Face] Figure 1 a provides the compound a 1013264167-0 10110847# in the Pane-1 cell at the end of 72 hours. Single number A〇1〇l Page 67 / Total 90 pages 201242597 A graph of the percent inhibition of single and combined doses of rotinib. Figure lb provides a graph of the percent inhibition of single and combined doses of Compound A and erlotinib in Panc-1 cells at the end of 96 hours. Figure 2a provides a graph of the percent inhibition of single and combined doses of Compound A and erlotinib in AsPc-1 cells at the end of 72 hours. Figure 2b provides a graph of the percent inhibition of single and combined doses of compound a with erlotinib in AsPc-1 cells at the end of 96 hours. Figure 3a provides a graph of ic5Q that determines compound a, erlotinib, lapatinib, and gemcitabine in Panc-1 cells. Figure 3b provides a graph of 1C that determines compound a, erlotinib, lapatinib, and gemcitabine in MiaPaca-2 cells.

0 U Figure 4a provides 30 single and combined dose percent cytotoxicity of gemcitabine (1C), compound 30 A (IC30), and erlotinib (IC3G) / lapatinib (1C) in Pane-1 cells The chart of the results. Figure 4b provides the percentage of single and combined doses of gemcitabine (ic), compound 5 0 A ( IC3 〇), and erlotinib (IC3()) / lapatinib (ic3()) in Pane-Ι cells. Figure of cytotoxicity results. Figure 5a provides single and combination of gemcitabine (1C), chemomerase A (ICqn), and erlotinib (ICQn) / lapatinib (ICon) in MiaPaca-2 cells. A graph of the percentage of cytotoxicity results. Figure 5b provides gemcitabine (1C) in MiaPaca-2 cells.

DU Compound A (IC30) and erlotinib (IC3()) / lapatinib (IC3() 10110847#单号A〇101 page 68/90 pages 201242597) single and combined dose percentage cells A chart of toxicity results. Figure 6 provides the percentage of single and combined doses of gemcitabine (ic or ic), compound 3 0 50 A (IC3〇 or 1C5〇), and erlotinib (IC3() or IC5()) in HPAC cells. A chart of cytotoxicity results. Figure 7 provides gemcitabine (1C or 1C), chemokine A (IC3() or IC5{)), and erlotinib (IC or 1 (:)) in CAPAN cells.

〇 U 〇 U I and a graph of the percentage cytotoxicity results for the combined doses. Figure 8a provides a graph of inhibition of pEGFR-Y1173 at 8 hours in combination with gemcitabine (ic) and compound A + erlotinib or compound A + lapatinib in Pane-1 cells. Figure 8b provides a graph of inhibition of pEGFR-Y845 at 8 hours in combination with gemcitabine (ic or 1C) and compound A + erlotinib or with compound A + lapatinib in Pane-Ι cells. Figure 8c provides a graph of inhibition of pAKT-S473 at 12 hours in combination with gemcitabine (ic or 1C) 3050 with compound A + erlotinib or with compound A + lapatinib in Pane-1 cells. Figure 8d provides a graph of inhibition of PRB-S780 at 12 hours in combination with gemcitabine (ic or 1C) Ο 30 50 and compound A + erlotinib or with compound A + lapatinib in Pane-Ι cells. Figure 8e provides a graph of inhibition of cyclin D at 12 hours in combination with gemcitabine (1C or 1C) 3 0 50 with compound A + erlotinib or with compound A + lapatinib in Pane-Ι cells. Figure 9 provides the activation of sulfur at 48 hours in combination with gemcitabine (IC3() or IC5()) and compound A + erlotinib or with compound A + lapatinib in Pane-Ι cells and MiaPaca-2 cells. Chart 101 of the cystine protease 3 7 dream list · A0101 Page 69 / Total 90 pages 1013264167-0 201242597 [Main component symbol description] [0031] No HH1084# Single number dish 01 Page 70 / Total 90 pages 1013264167- 0

Claims (1)

201242597 VII. Patent application scope: 1. A pharmaceutical combination f comprises a C D K inhibitor selected from the compound of formula I; OH 〇 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: A; nitro, cyano, C, -C alkyl , trifluoromethyl, hydroxy or ^-. An alkoxy group; or a pharmaceutically acceptable salt or solvate thereof; and a compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity. 2. The pharmaceutical combination according to claim 1, wherein in the compound of formula I, the phenyl group is substituted by 1, 2 or 3 identical or different substituents selected from the group consisting of: gas, bromine , fluoro or iodo, qc,-alkyl or trifluoroindolyl; or a pharmaceutically acceptable salt or solvate thereof. 3. The pharmaceutical combination of claim 2, wherein in the compound of formula I, the phenyl group is substituted with a gas. 4. The pharmaceutical combination according to claim 3, wherein the compound of formula I is (+)-trans-2-(2- gas-phenyl)-5,7-diylidene-8-( 2-Hydroxy-methyl-1-methyl-pyridyl-3-yl)-benzo-D-D--4-indole hydrochloride (Compound A). 5. The pharmaceutical combination according to claim 2; wherein in the compound of formula I 10110847#单号厅01第71页/90页1013264167-0 201242597, the phenyl group is replaced by two different Substituted substituents The two different substituents are selected from the group consisting of chlorine and trifluoromethyl. The pharmaceutical combination according to claim 5, wherein the compound of the formula I is (+)-trans-3-[2[(2- gas-4-trifluoromethyl-phenyl)-5. 7-Dihydroxy-8-(2-hydroxyindol-1-methyl-pyrrolidin-3-yl)- benzopiperel-4-one hydrochloride (Compound b). The pharmaceutical composition according to claim 1, wherein the compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is selected from the group consisting of erlotinib or lapatinib. The pharmaceutical composition according to claim 7, wherein the compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is erlotinib. The pharmaceutical composition according to claim 7, wherein the compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is lapatinib. 10. The pharmaceutical combination according to claim 1, wherein the group comprises gemcitabine according to the pharmaceutical combination described in claim 2; and further comprises gemcitabine. The pharmaceutical combination according to claim 4; wherein the combination further comprises gemcitabine. 13. The pharmaceutical combination of claim 6, wherein the combination further comprises gemcitabine. Gemcitabine is included as described in claim 7 of the scope of application. 15. The 10110847# single number A0101 further 11 12 as described in claim 1 wherein the combination is a pharmaceutical combination; wherein the combination is more medically combined; wherein the group is suitable for page 72 of 90 pages 1013264167-0 201242597 For the treatment of pancreatic cancer. The pharmaceutical combination according to claim 10; wherein the group is suitable for treating pancreatic cancer. 17. A method for treating pancreatic cancer in a body comprising administering to a subject a pharmaceutical effective amount of a CDK inhibitor in combination with a therapeutically effective amount of a compound capable of inhibiting EGFR kinase activity, The CDK inhibitor is selected from the group consisting of the compound of formula I; OH 〇 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: dentate; nitro, cyano, C, -C,-alkane a group, a trifluoromethyl group, a hydroxyalkoxy group; or a pharmaceutically acceptable salt or solvate thereof. 18. The method of claim 17, wherein in the compound of formula I, the phenyl group is substituted with a gas. 19. The method of claim 18, wherein the compound of formula I is (+)-trans-2-(2-a-phenyl)-5,7-dihydroxy-8-(2- Hydroxy-methyl_1_methyl_σ piroxime _3_yl)-benzo π-bottom _4_ hole hydrochloride (Compound A). 20. The method of claim 17, wherein the phenyl group is substituted by two different substituents in the formula 10110847#单号A_第73页/90 pages 1013264167-0 201242597. Substituted substituents, the two different substituents are selected from the group consisting of gas and trifluoromethyl. 21. The method of claim 20, wherein the compound of formula I is (+)-trans-3-[2[(2-chloro-4-trifluoromethyl-phenyl)-5, 7-Dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-benzopiperan-4-one hydrochloride (Compound B). The method of claim 17, wherein the compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is selected from the group consisting of erlotinib or lapatinib. The method of claim 22, wherein the compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is erlotinib 24, as in the method of claim 22, wherein The compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is lapatinib 25. A method for treating pancreatic cancer in a body comprising a medically effective amount of a CDK inhibitor Administration to a subject in combination with a therapeutically effective amount of a compound capable of inhibiting EGFR kinase activity selected from the group consisting of a compound of formula I; OH 〇 10110847^^'^^ A〇101 ^ 74 1 / * 90 I 1013264167-0 201242597 wherein Ar is a -phenyl group which is unsubstituted or substituted by a substituent which is the same or different, and the substituent is selected From: dentate; succinyl, aryl, W alkyl, trifluoromethyl, thiol or oxyalkyl; or a pharmaceutically acceptable salt or solvate thereof. The method of claim 25, wherein the phenyl group is substituted by a gas in the molecular phantom compound. 27. The method of claim 26, wherein the molecular formula is (1)-trans-2-(2-a-phenyl)-57-di-diyl 1 (2-dimethylmethyl-methyl) Called biting -3-yl) benzophenanthrene _4, hydrochloride ( 化合物 compound A). 28. The method of claim 25, wherein in the molecular phantom compound, the phenyl group is a substituent substituted by two different substituents selected from the group consisting of gas and trifluoro methyl. 29. The method of claim 28, wherein the molecular formula is (1)-trans-3-[2[(2-gas + Sanden methyl-phenyl)_5, dihydroxy-- 8-(2-carbylmethyl-bumethyl- cyano- _3_yl)-benzo-3-anthracene 4-one hydrochloride (compound. ◎ 30. The method described in claim 25, The compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is selected from the group consisting of erlotinib or lapatinib. The method of claim 30, wherein the epidermal growth factor is inhibited The compound of the EGFR kinase activity is erlotinib. The method of claim 30, wherein the compound capable of inhibiting epidermal growth factor receptor (EGFR) kinase activity is Rani. _847Bill No. _ Page 75 / Total 90 Page 1013264167-