HK40016954A - Combinations of akt and mek inhibitor compounds, and methods of use - Google Patents

Description

Combinations of AKT and MEK inhibitor compounds and methods of use thereof

The present application is a divisional application of a patent application having chinese application No. 201280027030.0 entitled "combination of AKT and MEK inhibitor compounds and methods of use thereof" filed on 3, 30, 2012 (PCT application No. PCT/US 2012/031716).

Cross Reference to Related Applications

This application claims priority to U.S. provisional application 61/471,038 filed on 1/4/2011. The entire contents of this provisional application are incorporated by reference into this application.

Technical Field

The present invention relates generally to pharmaceutical combinations of compounds that are active against hyperproliferative diseases such as cancer and which include compounds that inhibit AKT kinase activity. The invention also relates to methods of using said combinations for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells or associated pathological conditions.

Background

Protein Kinases (PKs) are enzymes that catalyze the phosphorylation of hydroxyl groups on tyrosine, serine, and threonine residues of proteins by transferring terminal (γ) phosphate on ATP. These enzymes regulate cell growth, differentiation and proliferation through signal transduction pathways, i.e., almost all aspects of The cell cycle are dependent on PK activity (Hardie, g. and Hanks, S. (1995) The protein kinase products book.i and II, Academic Press, San Diego, CA). In addition, abnormal PK activity has been implicated in a number of conditions, ranging from relatively non-life threatening diseases such as psoriasis to extremely fatal diseases such as glioblastoma (brain cancer). Protein kinases are an important target class for therapeutic regulation (Cohen, P. (2002) Nature rev. drug discovery 1: 309).

International patent application publication WO 2008/006040 discusses a number of AKT inhibitors, including the compound (S) -2- (4-chlorophenyl) -1- (4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ] pyrimidin-4-yl) piperazin-1-yl) -3- (isopropylamino) propan-1-one (formula I):

currently, there remains a need for improved methods and compositions that can be used to treat hyperproliferative diseases, such as cancer.

Disclosure of Invention

It has been determined that additive or synergistic effects in inhibiting the growth of cancer cells in vitro and in vivo can be achieved by administering a compound of formula I or a pharmaceutically acceptable salt thereof in combination with certain other specific drugs. The combinations and methods are useful for treating hyperproliferative diseases such as cancer.

Accordingly, certain embodiments of the present invention provide methods for treating a hyperproliferative disease in a mammal comprising administering to said mammal a combination of a compound of formula I:

in certain embodiments, the hyperproliferative disease is cancer.

In certain embodiments, the cancer is associated with a PTEN mutation.

In certain embodiments, the cancer is associated with AKT mutation, overexpression, or amplification.

In certain embodiments, the cancer is associated with a PI3K mutation.

In certain embodiments, the cancer is associated with Her2/ErbB2 amplification.

In certain embodiments, the cancer is selected from mesothelioma, endometrial, pancreatic, breast, lung, ovarian, prostate, melanoma, gastric, colon, renal, head and neck, and glioma.

In certain embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973, or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with PD-0325901, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of formula I or salt thereof is administered concurrently with the combination of the one or more agents.

In certain embodiments, the compound of formula I or salt thereof and the one or more agents are administered sequentially.

In certain embodiments, administration of the one or more agents begins about 1 to about 10 days prior to administration of the combination.

In certain embodiments, the compound of formula I or salt thereof is administered beginning about 1 to about 10 days prior to administration of the combination.

In certain embodiments, the compound of formula I or salt thereof and the one or more agents are administered beginning on the same day.

Certain embodiments of the present invention provide a compound of formula I, or a pharmaceutically acceptable salt thereof, for therapeutic use in improving the quality of life of a patient treated for a hyperproliferative disease, together with an agent selected from GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide methods for treating a disease or condition modulated by AKT kinase in a mammal, comprising administering to the mammal a) a compound of formula I or a pharmaceutically acceptable salt thereof; and b) one or more drugs selected from GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide a) a compound of formula I or a pharmaceutically acceptable salt thereof; and b) a combination of one or more drugs selected from the group consisting of GDC-0973 and PD-0325901 for use in the treatment of a hyperproliferative disease.

Certain embodiments of the present invention provide a) a compound of formula I or a pharmaceutically acceptable salt thereof; and b) a combination of one or more drugs selected from GDC-0973 and PD-0325901 for use in the treatment of a disease or disorder modulated by AKT kinase.

Certain embodiments of the present invention provide the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with GDC-0973 and PD-0325901, in the manufacture of a medicament for the treatment of a hyperproliferative disease in a mammal.

Certain embodiments of the present invention provide the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, in combination with GDC-0973 and PD-0325901, in the manufacture of a medicament for treating a disease or condition modulated by AKT kinase in a mammal.

Certain embodiments of the present invention provide kits for treating hyperproliferative disorders comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, a container, and a package insert or label indicating administration of the compound of formula I with one or more drugs selected from GDC-0973 and PD-0325901.

Certain embodiments of the present invention provide products comprising a compound having formula I or a pharmaceutically acceptable salt thereof and one or more agents selected from GDC-0973 and PD-0325901; the product is used as a combined preparation for separate, simultaneous or sequential use in the treatment of hyperproliferative diseases.

Synergistic/additive effects were observed when the combination of GDC-0068 and GDC-0973 was administered in vitro in a number of cell types including melanoma, lung, colon, ovarian, renal, breast, prostate, pancreatic cancer cell lines, and these findings have been demonstrated in vivo in melanoma, colon, and lung cancer xenograft models. Synergistic effects were observed in tumor types driven by Ras/Raf or activation of both pathways. When the combination of GDC-0068 and GDC-0973 was administered in a variety of cells, a synergistic effect was shown in melanoma, lung cancer (e.g., NSCLC), and colon cancer cell lines. Breast cancer cells, including luminal (ER +), Her2+, and basal triple negative breast cancers, may also show synergistic effects when given a combination of GDC-0068 and GDC-0973. When the combination of GDC-0068 and GDC-0973 was administered, a synergistic effect was observed even in cells sensitive to Meki alone.

The mutation status of cancer cells has been found to be a biomarker of how the cancer cells respond to different treatment regimens. For example, cancer cells having a PI3K pathway (e.g., PI3K or AKT) mutation in combination with a Kras and/or Braf mutation can show a positive (e.g., synergistic) response to a combination therapy described herein. Furthermore, the PTEN status of cancer cells is also a biomarker. Thus, certain embodiments of the invention include methods of using these combination therapies to treat cancer cells (in vitro or in vivo) having a combination of these biomarkers. Certain embodiments of the invention include selecting patients for combination therapy with a combination of these biomarkers.

A strong synergistic effect of the combination of GDC-0068 and GDC-0973 was observed in the A2058(PTEN No/Braf mutant) melanoma model. Comparable single drug Tumor Growth Inhibition (TGI) was observed at all doses of GDC-0973 and at the higher doses of GDC-0068 (75 and 100 mg/kg). TGI was not observed in 50mg/kg of GDC-0068. The combination of both drugs was well tolerated in this model with a maximum weight loss of-13%.

In addition to providing improved treatment for a given hyperproliferative disease, administration of certain combinations of the invention may improve the quality of life of a patient (as compared to the quality of life experienced by the same patient receiving a different treatment). For example, administration of a combination of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a medicament described herein to a patient may provide improved quality of life (as compared to the quality of life that the same patient would experience if the patient received only chemotherapeutic agents as treatment). For example, combination therapy using the combinations described herein can reduce the dosage of therapeutic agent needed, thereby attenuating the side effects associated with high doses of chemotherapeutic agents (e.g., nausea, vomiting, hair loss, rash, loss of appetite, weight loss, etc.). The combination may also cause reduced tumor burden and associated adverse events such as pain, organ dysfunction, weight loss, and the like. Accordingly, one aspect of the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for therapeutic use in conjunction with a medicament as described herein to improve the quality of life of a patient for the treatment of a hyperproliferative disease.

Drawings

FIG. 1 illustrates the results of the combination of GDC-0068 and GDC-0973 (2.5mg/kg) versus tumor volume.

FIG. 2 illustrates the results of the combination of GDC-0068 and GDC-0973 (5.0mg/kg) versus tumor volume.

FIG. 3 illustrates the results of the combination of GDC-0068 and GDC-0973(7.5mg/kg) versus tumor volume.

FIG. 4 illustrates the results of a combination of GDC-0068 and GDC-0973 on in vitro colorectal cancer cell lines.

FIG. 5 illustrates the results of the combination of GDC-0068 and GDC-0973 on HCT-116 (colon cancer-PI 3K and Kras mutant), i.e., the results of Mek and Akt in vitro combination of HCT-116 (colon-PI 3K and Kras mutation). A two-dimensional (2D) heat map (heatmap) showing the combined effect on cell survival of HCT-116 cells is shown. The increasing concentration of GDC-0068 is shown on the x-axis and the increasing concentration of GDC-0973 is shown on the y-axis. On the right side, a percentage inhibition (% inhibition) heat map is shown indicating the percentage inhibition of GDC-0068 and GDC-0973 at each concentration in combination or as single drug; the vehicle (DMSO) exposure control was set to 0. BLISS scores were calculated for each dose pair and heat map shown on the left.

FIG. 6 illustrates the results of a combination of GDC-0068 and GDC-0973 on NSCLC cell lines in vitro.

FIG. 7 illustrates the results of the combination of GDC-0068 and GDC-0973 on H2122(NSCLC-Kras mutant), i.e.NSCLC-Kras. Two-dimensional (2D) heatmaps show the combined effect on cell survival of NCI-H2122 cells, i.e., the results of the in vitro combination of Mek and Akt of H2111(NSCLC-Kras mutation). The increasing concentration of GDC-0068 is shown on the x-axis and the increasing concentration of GDC-0973 is shown on the y-axis. On the right side, a percentage inhibition (% inhibition) heat map is shown indicating the percentage inhibition of GDC-0068 and GDC-0973 at each concentration in combination or as single drug; the vehicle (DMSO) exposure control was set to 0. BLISS scores were calculated for each dose pair and heat map shown on the left.

FIG. 8 illustrates the results of a combination of GDC-0068 and GDC-0973 on melanoma cell lines in vitro.

FIG. 9 illustrates the results of a single drug and the combination of GDC-0068 and GDC-0973 on A2058 (melanoma-PTEN-/-and Braf mutants), i.e., the results of the in vitro combination of Mek and Akt of A2058 (melanoma-PTEN-/-and Braf mutations) with minimal in vitro single drug activity. Two-dimensional (2D) heatmaps are shown showing the combined effect on cell survival of a2058 cells. BLISS scores were calculated for each dose pair and heat map shown on the left. The increasing concentration of GDC-0068 is shown on the x-axis and the increasing concentration of GDC-0973 is shown on the y-axis. On the right side, a percentage inhibition (% inhibition) heat map is shown indicating the percentage inhibition of GDC-0068 and GDC-0973 at each concentration in combination or as single drug; the vehicle (DMSO) exposure control was set to 0.

Figure 10 illustrates the enhanced knock-down effect (knockdown) of AKT and MEK pathway activity compared to single drug.

FIG. 11 illustrates the results of a combination of GDC-0973 and GDC-0068 against MDA-MB-468 breast cancer cell line.

FIG. 12 illustrates the results of a combination of GDC-0068 and GDC-0973 on in vitro breast cancer cell lines.

FIG. 13 illustrates the results of a combination of GDC-0068 and GDC-0973 on ovarian cancer.

FIG. 14 illustrates the results of a combination of GDC-0068 and GDC-0973 on in vitro prostate cancer cell lines.

FIG. 15 illustrates the results of an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor) in MX-1 breast tumors.

FIG. 16 illustrates the results of an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor) in H2122 NSCLC tumors.

FIG. 17 illustrates the results of an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor) in SW1990 pancreatic tumors.

FIG. 18 illustrates the results of an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor) in Pa _ Tu-8902 pancreatic tumors.

FIG. 19 illustrates the results in 537Mel for an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor).

FIG. 20 illustrates the results of an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor) in A2058 melanoma.

FIG. 21 illustrates the results of an orally administered combination of GDC-0068+ GDC-0973(MEK inhibitor) in HCT-116 colorectal tumors.

FIGS. 22a-22b show results comparing inhibition of cell survival of various cell lines by single agents and combination therapies. GDC-0068 cellular potency correlates with Akt activation by alteration of PI3K/PTEN/HER2, while GDC-0973 cellular potency correlates with MEK activation by RAS or B-RAF mutations. GDC-0068-and GDC-0973-sensitive cell lines are generally mutually exclusive. About one third of the tested cell lines showed resistance to both drugs (see FIGS. 22a-22 b). In most of the cell lines tested, the combination of GDC-0068 and GDC-0973 resulted in enhanced inhibition of cell survival compared to the single drug used alone. The combined effect was evaluated using the BLISS-independent model (Leh a-r etal.2007).

FIG. 22a is a graphic illustration on single drug IC50 for GDC-0068 and GDC-0973 in multiple cancer cell lines. Cells were treated with increasing concentrations of GDC-0068 or GDC-0973 or in combination with RPMI + 10% FBS and assayed for viability after 4 days using CelTiter-Glo. The corresponding lower panel illustrates the synergistic effect of the combination of GDC-0068 and GDC-0973 against several specific genotypes. Colored modules represent mutations, deletions or activations. Mutations/alterations of B-RAF, RAS, HER2, PI3K or PTEN are indicated by colored squares under each cell line (B-RAF, brown; RAS, red; HER2, blue; PTEN, dark green; for PI3K, light green indicates a kinase domain mutation of PIK3CA, light blue indicates a mutation or amplification of the non-kinase domain). PTEN alteration indicates a signal or gene mutation that cannot be detected by protein spotting for this protein. The tissue origin for each cell line is also indicated by different colors and letters, i.e., breast cancer (Br), colon cancer (Co), non-small cell lung cancer (Lu), melanoma (Me), ovarian cancer (Ov), prostate cancer (Pr), and renal cancer (Re).

FIG. 22b illustrates the overall positive combined Bliss scores in a multiplex cell line for GDC-0068 and GDC-0973. Synergistic effects were observed in multiple cell lines, as indicated by overall positive BLISS scores, particularly in cell lines with RAS/RAF pathway activation or in cell lines with PI3K/Akt and RAS/RAF pathway activation.

An overall positive BLISS score was calculated from the combination of GDC-0068 and GDC-0973 in each cell line.

FIG. 23 illustrates the Bliss heat map and% inhibition in 537MEL melanoma (PTEN none, Braf amp/del) for GDC-0068 and GDC-0973; the combination of GDC-0068 and GDC-0973 inhibits both pathways and increases cell death.

FIG. 24 illustrates protein spot analysis for human HTC116 colon cancer cell line treated with GDC-0068 and GDC-0973 cell lines for 24 hours. HCT-116 cells were cultured with specific concentrations of GDC-0068 and GDC-0973 for about 3 hours. Phosphorylation of Akt, MEK and their downstream markers was analyzed by protein spotting.

FIG. 25 illustrates that GDC-0068 and GDC-0973 in combination increase the effect in 537MEL melanoma (PTEN none, Braf amp/del).

Figure 26 illustrates significant changes in phosphoprotein expression levels for the combination of GDC-0068 and GDC-0973 relative to vehicle control. A2058x1 tumors were collected 3 hours after administration of a single dose of GDC-0068(100mg/kg) or GDC-0973(7.5mg/kg) or combination to mice. Tumors were analyzed using Reverse Phase Protein Array (RPPA).

Figure 27 illustrates a significant change in phosphoprotein expression levels for the combination of GDC-0068 and GDC-0973 relative to single agents after administration in a2058 xenograft tumors.

Figure 28 illustrates a two-dimensional (2D) heat map showing the combined effect on cell survival of MALME3M cells. The increasing concentration of GDC-0068 is shown on the x-axis and the increasing concentration of GDC-0973 is shown on the y-axis. Percent inhibition (% inhibition) heatmap shows the percent inhibition of GDC-0068 and GDC-0973 at each concentration in combination or as a single drug; the vehicle (DMSO) exposure control was set to 0.

Figure 29 illustrates a two-dimensional (2D) heat map showing the combined effect on cell survival of MALME3 cells. The increasing concentration of GDC-0068 is shown on the x-axis and the increasing concentration of GDC-0973 is shown on the y-axis. Percent inhibition (% inhibition) heatmap shows the percent inhibition of GDC-0068 and GDC-0973 at each concentration in combination or as a single drug; the vehicle (DMSO) exposure control was set to 0. BLISS scores were calculated for each dose pair and heat map shown on the right.

Figure 30 illustrates a two-dimensional (2D) heat map showing the combined effect on cell survival of NCI-BL2122 cells. The increasing concentration of GDC-0068 is shown on the x-axis and the increasing concentration of GDC-0973 is shown on the y-axis. Percent inhibition (% inhibition) heatmap shows the percent inhibition of GDC-0068 and GDC-0973 at each concentration in combination or as a single drug; the vehicle (DMSO) exposure control was set to 0. BLISS scores were calculated for each dose pair and heat map shown on the right.

FIG. 31 shows changes in phosphoprotein expression levels (24 hours) and the modulation of AKT and MEK pathways using a combination of GDC-0068 and GDC-0973. A2058x1 tumors were collected 24 hours after administration of a single dose of GDC-0068(100mg/kg) or GDC-0973(7.5mg/kg) or a combination to mice. Tumors were analyzed using Reverse Phase Protein Array (RPPA).

Detailed Description

The words comprises/comprising when used in this specification and claims are to specify the presence of stated features, integers, components or steps but they do not preclude the presence or addition of one or more other features, integers, components, steps or groups thereof.

The terms "treatment" and "treatment" refer to both therapeutic treatment and prophylactic measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development or spread of cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "treatment" may also mean an extended survival compared to the expected survival without treatment. Subjects in need of treatment include subjects already suffering from a condition or disorder as well as subjects susceptible to such a condition or disorder or subjects in whom such a condition or disorder should be prevented.

The phrase "therapeutically effective amount" means (i) an amount of a compound of the invention that treats or prevents a particular disease, condition, or disorder described herein, (ii) an amount of a compound of the invention that attenuates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder described herein, or (iii) an amount of a compound of the invention that prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with cancer. A drug may be cytostatic (cytostatic) and/or cytotoxic if it can prevent the growth of cancer cells and/or kill existing cancer cells. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR).

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma (carcinoma), lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung (adenocarcinosoma of the lung) and squamous carcinoma of the lung (squamous carcinosoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric cancer (gastric or stomachma cancer) including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, hepatoma (hepatoma), breast cancer (breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer (vulval cancer), thyroid cancer, hepatic carcinoma (hepatic carcinosoma), anal cancer, penile cancer, and head and neck cancer. Gastric cancer, as used herein, includes gastric cancer (stomachs), which may develop in any part of the stomach and may spread throughout the stomach and to other organs; in particular the oesophagus, the lung, the lymph nodes and the liver.

"chemotherapeutic agents" are biological (macromolecular) or chemical (small molecule) compounds used to treat cancer (independent of mechanism of action).

A "platinum agent" is a chemotherapeutic agent that includes platinum, such as carboplatin, cisplatin, and oxaliplatin.

The term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs, sheep, and livestock. The term patient refers to a mammal, and in one embodiment, the patient is a human.

The term "package insert" is used to refer to instructions generally included in the commercial packaging of therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

The phrase "pharmaceutically acceptable salt" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate (methanesulfate) "methanesulfonate (mesylate)", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1' -methylene-bis (2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts can be contemplated to comprise another molecule, such as an acetate, succinate, or other counterion. The counterion can be any organic or inorganic moiety (moiety) that stabilizes the charge on the parent compound. Moreover, a pharmaceutically acceptable salt may have more than one charged atom in its structure. The plurality of charged atoms in the case of a pharmaceutically acceptable salt can have a plurality of counterions. Thus, pharmaceutically acceptable salts can have one or more charged atoms and/or one or more counterions.

The desired pharmaceutically acceptable salts can be prepared by any suitable method available in the art. For example, the free base is treated with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like, or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosyl acid (glucuronic acid) such as glucuronic acid or galacturonic acid, alpha-hydroxy acid such as citric acid or tartaric acid, amino acid such as aspartic acid or glutamic acid, aromatic acid such as benzoic acid or cinnamic acid, sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, and the like. Acids which are generally considered suitable for forming pharmaceutically acceptable salts from basic Pharmaceutical compounds are discussed, for example, in P.Stahl et al, Camile G. (eds.) Handbook of Pharmaceutical salts. Berge et al, Journal of Pharmaceutical Sciences (1977)66(1) 119; gould, International J.of pharmaceuticals (1986) 33201217; anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; remington's Pharmaceutical Sciences,18^thed., (1995) Mack Publishing Co., Easton PA; and The Orange Book (Food)&Drug Administration, Washington, d.c. on the third website). These disclosures are incorporated by reference into this application.

The phrase "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and/or toxicologically with the other ingredients comprising the formulation and/or the mammal to be treated.

As used herein, "synergistic effect" refers to a more effective therapeutic combination compared to the additive effects of two or more single drugs. The synergistic interaction between a compound of formula I or a pharmaceutically acceptable salt thereof and one of GDC-0973 and PD-0325901 can be determined from the results obtained from the assays described herein. The results of these assays can be analyzed using the Chou and Talalay combination method and dose-response analysis using CalcuSyn software to obtain a combination index (Chou and Talalay, 1984, adv. enzyme Regul.22: 27-55). The combinations provided herein have been evaluated in several assay systems, and the data can be analyzed using standard procedures for quantifying synergistic, additive, and antagonistic effects between anti-cancer agents. Illustrative examples are described in Chou and Talalay, in "New Avenues in development Cancer Chemotherapy," Academic Press,1987, Chapter 2. A combination index of less than 0.8 indicates a synergistic effect, a value of greater than 1.2 indicates antagonism and a value between 0.8 and 1.2 indicates an additive effect. The combination therapy may provide "synergy" and demonstrate a "synergistic effect", i.e. the effect achieved when the active ingredients are used together is greater than the sum of the effects obtained from the separate use of the compounds. The synergistic effect can be obtained under the following conditions: (1) when the active ingredients are co-formulated in a combined unit dose formulation and administered or delivered simultaneously; (2) when the active ingredients are delivered alternately or in parallel as separate formulations; or (3) when passing through some other scheme. When delivered in alternation therapy, a synergistic effect may be obtained when the compounds are administered or delivered sequentially, e.g. by different injections with separate syringes. In general, an effective dose of each active ingredient is administered sequentially, i.e., consecutively, during alternation therapy, while in combination therapy, effective doses of two or more active ingredients are administered together. The combined effect was evaluated using the BLISS-independent model as well as the highest single drug (HSA) model (Leh a r et al 2007, Molecular Systems Biology 3: 80). The degree of potentiation of a single drug was quantified by the BLISS score and a positive BLISS score (greater than 0) indicated greater than simple accumulation. A cumulative positive BLISS score greater than 250 is considered a strong synergistic effect observed over the range of concentrations tested. The HAS score (greater than 0) indicates that the combined effect is greater than the maximum single drug response at the corresponding concentration.

One aspect includes a method of Tumor Growth Inhibition (TGI) in a patient having a cancer that includes PI3K, AKT, or PTEN mutations, and which further includes RAS/RAF mutations in one example, comprising administering to the patient GDC-0068 and one of GDC-0973 and PD-0325901, or a pharmaceutically acceptable salt thereof. In certain embodiments, the combination is synergistic. In certain embodiments, the TGI of the combination is greater than the TGI of GDC-0068 or one of GDC-0973 and PD-0325901 alone. In certain embodiments, the TGI of the combination is greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% of the TGI of GDC-0068 or one of GDC-0973 and PD-0325901, alone.

Methods of measuring TGI are known in the art. In one exemplary method, the mean tumor volume is determined and a comparison is made between patients before and after treatment. Tumor volume can be measured in two dimensions (length and width) using any method in the art such as UltraCal IV caliper (Fred v. fowler Company) or via PET (positron emission tomography) or some other method. The formula can be used: tumor volume (mm)³) Length x width²) x is 0.5. Measuring tumor volume over multiple time periods may be accomplished using the Linear Mixing Effect (LME) method of mixing modeling (Pinheiro et al 2009). The method can address repeated measurements (and multiple patients). Cubic regression splines can be used to fit the non-linear distribution to the time course of tumor volume at each dose level. These non-linear distributions can then be correlated to the dose within the mixture model. Tumor growth inhibition as a percentage of vehicle can be calculated as follows: percent area under the curve fitted per day (AUC) related to vehicle using the following formula:

using this formula, a TGI value of 100% indicates tumor stasis, a TGI value greater than about 1% but less than about 100% indicates tumor growth inhibition, and a TGI value greater than about 100% indicates tumor regression.

Preparation of Compounds of formula I

The compounds of formula I and their salts may be prepared as described in international patent application publication WO 2008/006040 or as described in example 1 below. In preparing compounds of formula I, it may be desirable to protect remote functional groups (e.g., primary or secondary amines, etc.) of intermediates. The need for such protection will vary depending on the nature of the distal functional group and the conditions of the preparation method. Suitable amino-protecting groups (NH-Pg) include acetyl, trifluoroacetyl, tert-Butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection can be readily determined by one skilled in the art. For a general description of protecting Groups and their use, see t.w. greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

Separation method

In any synthesis process for the preparation of compounds of formula I, it is advantageous to separate the reaction products from each other and/or from the starting materials. The desired product of each step or series of steps is isolated and/or purified to a target homogeneity using techniques common in the art. Typically, such separation includes heterogeneous extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography. Chromatography may include a number of methods including, for example: reverse phase and normal phase chromatography; volume exclusion chromatography; ion exchange chromatography; high, medium and low pressure liquid chromatography and apparatus; small-scale analysis; simulated Moving Bed (SMB) and preparative thin layer chromatography or thick layer chromatography, as well as small scale thin layer and flash chromatography techniques.

Another separation method comprises: the reaction mixture is treated with a reagent selected to bind to or separate the desired product, unreacted starting materials, reaction by-products, and the like. Such agents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, and the like. Alternatively, the reagent may be an acid (in the case of a basic material), a base (in the case of an acidic material), a binding reagent such as an antibody, a binding protein, a selective chelator such as a crown ether, a liquid/liquid ion extraction reagent (LIX), or the like.

The selection of a suitable separation method depends on the nature of the substances involved. For example, boiling point and molecular weight in distillation and sublimation, presence or absence of polar functional groups in chromatography, substance stability in acidic and basic media in heterogeneous extraction, and the like. The skilled person will apply the most suitable technique to achieve the desired separation.

Mixtures of diastereomers may be separated into their individual diastereomers on the basis of physicochemical differences by methods well known to those skilled in the art, such as chromatography and/or fractional crystallization. Enantiomers can be separated as follows: the enantiomeric mixtures are converted into diastereomeric mixtures by reaction with a suitable optically active compound (e.g., a chiral auxiliary, such as a chiral alcohol or Mosher's acid chloride), the diastereomers are separated, and the single diastereomers are converted (e.g., hydrolyzed) to the corresponding pure enantiomers. Also, some of the compounds of the present invention may be atropisomers (e.g., substituted biaryls) and are considered as part of the present invention. Enantiomers can also be separated using a chiral HPLC column.

Single stereoisomers, such as enantiomers (substantially free of their stereoisomers) may be obtained by resolution of a racemic mixture using, for example, a method of forming diastereomers using an optically active resolving agent. (Eliel, E.and Wilen, S. "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994; Lochmuller, C.H., J.Chromatogr., 1975)113(3): 283-. The racemic mixture of chiral compounds of the present invention is separated and isolated by any suitable method, which comprises: (1) forming ionic, diastereomeric salts with chiral compounds and separating by fractional crystallization or other means, (2) forming diastereomeric compounds with chiral derivatizing agents, separating the diastereomers and converting to pure stereoisomers, and (3) directly separating the substantially pure or enriched stereoisomers under chiral conditions. See: "Drug Stereochemistry, analytical methods and Pharmacology," Irving W.Wainer, Ed., Marcel Dekker, Inc., New York (1993). In process (1), diastereomeric salts can be formed from the reaction of an enantiomerically pure chiral base, e.g., brucine, quinine, ephedrine, strychnine, α -methyl- β -phenylethylamine (amphetamine), etc., with an asymmetric compound bearing an acidic functional group, e.g., carboxylic acid and sulfonic acid. Separation of diastereomeric salts can be induced by fractional crystallization or ion chromatography. For the separation of the optical isomers of amino compounds, the addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid or lactic acid, can lead to the formation of diastereomeric salts.

Alternatively, by process (2), the substrate to be resolved is reacted with one enantiomer of a chiral compound to form a diastereomer pair (E.and Wilen, S. "Stereochemistry of Organic Compounds", John Wiley&Sons, inc.,1994, p.322). Diastereomeric compounds can be formed by reaction of an asymmetric compound with an enantiomerically pure chiral derivatizing agent, such as a menthyl derivative, followed by separation of the diastereomers and hydrolysis to produce the pure or enriched enantiomers. The method for determining optical purity comprises: chiral esters of racemic mixtures, e.g., menthyl esters, e.g., (-) menthyl chloroformate, or Mosher esters (α -methoxy- α - (trifluoromethyl) phenyl acetate (Jacob III.J. org.chem., (1982)47:4165) are prepared in the presence of a base and analyzed¹H NMR spectroscopy, to determine the presence of two atropisomer enantiomers or diastereomers. The stable diastereoisomers of the atropisomeric compounds can be separated and isolated by normal and reverse phase chromatography according to the method for isolating the atropisomeric naphthyl-isoquinoline (WO 96/15111). By method (3), racemic mixtures of two enantiomers can be separated by Chromatography using a Chiral stationary phase ("Chiral Liquid Chromatography" (1989) W.J. Lough, Ed., Chapman and Hall, New York; Okamoto, J.of Chromatography. (1990)513: 375-. Enriched or pure enantiomers can be distinguished by methods used to distinguish other chiral molecules (with asymmetric carbon atoms), such as optical spin and circular dichroism.

Chemotherapeutic agents

Certain chemotherapeutic agents have proven surprising and unexpected properties for inhibiting cell proliferation in vitro and in vivo in combination with a compound of formula I or a pharmaceutically acceptable salt thereof. The chemotherapeutic agents include GDC-0973 and PD-0325901.

GDC-0973, also known as XL-518, is a selective inhibitor of MEK, also known as cytokinin-activated protein kinase (MAPKK), a key component of the RAS/RAF/MEK/ERK pathway that is frequently activated in human tumors. Inappropriate activation of the MEK/ERK pathway promotes cell growth in the absence of exogenous growth factors. A phase I clinical trial is underway to evaluate GDC-0973 against solid tumors. GDC-0973 can be prepared as described in international patent application publication WO2007044515(a 1). GDC-0973 has the following nomenclature: (S) - (3, 4-difluoro-2- (2-fluoro-4-iodophenylamino) phenyl) (3-hydroxy-3- (piperidin-2-yl) azetidin-1-yl) methanone, and having the structure:

PD-0325901(CAS registry No. 391210-10-9, Pfizer) is a second generation non-ATP competitive allosteric MEK inhibitor that is used for potential oral tablet therapy of cancer (US 6960614; US 6972298; US 2004/147478; US 2005/085550). Phase II clinical trials have been conducted for potential treatment of breast cancer tumors, colon cancer tumors, and melanoma. PD-0325901 is named (R) -N- (2, 3-dihydroxypropoxy) -3, 4-difluoro-2- (2-fluoro-4-iodophenylamino) benzamide and has the following structure:

pharmaceutical composition

The pharmaceutical compositions or formulations of the present invention comprise a compound of formula I or a pharmaceutically acceptable salt thereof, a chemotherapeutic agent, and a combination of one or more pharmaceutically acceptable carriers, glidants, diluents, or excipients.

One example includes a first formulation for oral delivery of GDC-0068 or a salt thereof and one or more pharmaceutically acceptable carriers, glidants, diluents, or excipients, and a second formulation for oral delivery of one or a salt of GDC-0973 and PD-0325901 and one or more pharmaceutically acceptable carriers, glidants, diluents, or excipients. In one example, the second formulation comprises GDC-0973 or a salt thereof.

The compound of formula I or pharmaceutically acceptable salts thereof and the chemotherapeutic agent may exist in unsolvated forms or solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and the present invention is intended to encompass both solvated as well as unsolvated forms.

The compounds of formula I or pharmaceutically acceptable salts thereof and chemotherapeutic agents may also exist in different tautomeric forms, all of which are included within the scope of the invention. The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that can interconvert through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions by migration of protons, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions through the reconstruction of some of the bonding electrons.

Pharmaceutical compositions encompass both bulk (bulk) compositions and single dose units comprising more than one (e.g., two) pharmaceutically active agents, including a compound of formula I or a pharmaceutically acceptable salt thereof and a chemotherapeutic agent as described herein, as well as any pharmaceutically inactive excipients, diluents, carriers, or glidants. The bulk composition, as well as each single dosage unit, may contain a fixed amount of the pharmaceutically active agent previously described. The bulk composition is a substance that has not been formed into a single dosage unit. Exemplary dosage units are oral dosage units such as tablets, pills, capsules, and the like. Similarly, the methods of treating a patient by administering a pharmaceutical composition of the invention described herein are also intended to encompass administration of bulk compositions and single dosage units.

Pharmaceutical compositions also encompass isotopically labeled compounds, which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. All isotopes of any particular atom or element recited are encompassed by the inventionCompounds and uses thereof. Exemplary isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, for example²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³²P、³³P、³⁵S、¹⁸F、³⁶Cl、¹²³I and¹²⁵I. certain isotopically-labelled compounds of the invention (e.g. by³H and¹⁴c-labeled) can be used in compound and/or substrate tissue distribution assays. Tritiated (i.e., tritiated) due to its ease of preparation and detection³H) And carbon-14 (i.e.¹⁴C) Isotopes are useful. In addition, with heavier isotopes such as deuterium (i.e. deuterium)²H) Substitution of (a) may provide certain therapeutic advantages because it results in greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and thus may be preferred in some circumstances. Positron emitting isotopes such as¹⁵O、¹³N、¹¹C and¹⁸f can be effectively used in Positron Emission Tomography (PET) studies to detect substrate receptor occupancy.

The compounds of formula I or pharmaceutically acceptable salts and chemotherapeutic agents thereof are formulated in accordance with standard pharmaceutical practice for therapeutic combinations of hyperproliferative diseases in mammals, including humans, for therapeutic treatment, including prophylactic treatment. The present invention provides pharmaceutical compositions comprising a compound of formula I or a pharmaceutically acceptable salt thereof in combination with one or more chemotherapeutic agents as described herein and one or more pharmaceutically acceptable carriers, glidants, diluents, or excipients.

Suitable carriers, diluents and excipients are well known to those skilled in the art and include such materials as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient employed will depend upon the method of use and the purpose for which the compounds of the present invention are to be used. The choice of solvent is generally based on solvents that one of skill in the art would consider safe (GRAS) to administer to a mammal. Generally, safe solvents are non-toxic aqueous solvents such as water, as well as other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300), and the like, and mixtures thereof. The formulations may also contain one or more buffering agents, stabilizing agents, surfactants, wetting agents, lubricants, emulsions, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colors, sweeteners, flavorants, flavoring agents and other known additives to provide a drug with an elegant appearance (i.e., a compound of the present invention or pharmaceutical composition thereof) or to aid in the preparation of a pharmaceutical product (i.e., a drug).

The formulations may be prepared using conventional dissolution and mixing methods. For example, the bulk drug substance (i.e., a compound of the invention or a stabilized form of the compound (e.g., a complex with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more excipients as described above. The compounds of the present invention are typically formulated in pharmaceutical dosage forms to provide an easily controlled dose of the drug and to enable patient compliance with prescribed dosing regimens.

The pharmaceutical compositions (or formulations) of the present application can be packaged in a variety of ways, depending on the method used to administer the drug. For example, the article for dispensing may comprise a container in which the pharmaceutical formulation is placed in a suitable form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders and the like. The container may also include a tamper-proof system to prevent inadvertent use of the contents of the package. In addition, the container has a label disposed thereon that describes the contents of the container. The tag may also include a suitable warning.

Pharmaceutical formulations of the compounds may be prepared for various routes and types of administration. For example, a compound of formula I, or a pharmaceutically acceptable salt thereof, having a desired purity can be optionally mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1995)18th edition, Mack publication. The formulations may be prepared by mixing at ambient temperature, at an appropriate pH, and in the desired purity, with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to recipients at the dosages and concentrations employed. The pH of the formulation depends primarily on the particular use and concentration of the compound, but ranges from about 3 to about 8.

The pharmaceutical formulation is optionally sterile. In particular, formulations for in vivo administration must be sterile. The sterilization can be easily accomplished by filtration through sterile filtration membranes.

Pharmaceutical formulations can generally be stored as solid compositions, lyophilized formulations, or aqueous solutions.

Pharmaceutical formulations are quantified and administered in a manner such that the amount, concentration, schedule, history, carrier and route of administration are in accordance with good medical practice. Factors considered in this application include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to a medical professional. The "therapeutically effective amount" of the compound administered depends on such considerations and is the minimum amount required to prevent, ameliorate or treat the coagulation factor-mediated condition. The amount is preferably below that which is toxic to the host or which renders the host significantly more prone to bleeding.

Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to the subject at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexa-hydrocarbonic quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gels, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamicAn amino amide, an aspartic acid, a histidine, an arginine, or a lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN^TM,PLURONICS^TMOr polyethylene glycol (PEG). The active pharmaceutical ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gel-microcapsules and poly (methylmethacylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively. This technique is disclosed in Remington's pharmaceutical sciences 18th edition, (1995) Mack publication.

Sustained release formulations of the compounds of formula I or pharmaceutically acceptable salts thereof may be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing a compound of formula I, or a pharmaceutically acceptable salt thereof, in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM((injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly D- (-) -3-hydroxybutyric acid.

Pharmaceutical formulations include those suitable for the route of administration detailed herein. The formulations may suitably be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally available in Remington's pharmaceutical Sciences 18^thEd. (1995) Mack Publishing co., Easton, PA. The method comprises the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by the following methodPreparing: so that the active ingredient is uniformly and intimately associated with a liquid carrier or a finely divided solid carrier or both and, if necessary, shaping the product.

Formulations of a compound of formula I or a pharmaceutically acceptable salt thereof and/or a chemotherapeutic agent suitable for oral administration may be prepared as discrete units such as a pill, hard or soft capsule (e.g. gelatin capsule), cachet, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, syrup or spirit each containing a predetermined amount of a compound of formula I or a pharmaceutically acceptable salt thereof and/or a chemotherapeutic agent. As a combined preparation, the amount of the compound of formula I or a pharmaceutically acceptable salt thereof and the amount of the chemotherapeutic agent may be formulated as a pill, capsule, solution or suspension. Alternatively, the compound of formula I or a pharmaceutically acceptable salt thereof and the chemotherapeutic agent may be separately formulated as a pill, capsule, solution or suspension for alternate administration.

The formulations may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a palatable preparation. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

The tablets may optionally be coated or scored and optionally formulated so as to provide slow or controlled release of the active ingredient therefrom. Tablet excipients for pharmaceutical formulations may include: a filler (or diluent) that increases the total volume of the powdered drug constituting the tablet; disintegrants, which facilitate disintegration of the tablet into small fragments, ideally single drug particles, upon ingestion and facilitate rapid dissolution and absorption of the drug; a binder which ensures that the granules and tablets can be formed after compression with the required mechanical strength and holds the tablets together, which prevents them from breaking apart into their component powders during packaging, shipping and normal handling; a glidant which improves the flowability of the powder constituting the tablet during production; lubricants which ensure that the tableting powder does not adhere to the apparatus used to compress the tablet during manufacture, which improve the flow of the powder mixture through the press and minimise friction and breakage as the finished tablet is ejected from the apparatus; antiadherents, having a function similar to glidants, which reduce the adhesion between the powder constituting the tablet and the machine used to compress the tablet during manufacture; flavors incorporated into the tablets to provide a suitable taste or to mask an undesirable taste; and colorants to aid in identification and patient compliance.

Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets are acceptable. These excipients may be, for example, inert diluents such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; and lubricating agents such as magnesium stearate, stearic acid or talc. Tablet formulations may be uncoated or coated by known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

For the treatment of the eye or other external tissues such as mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075-20% w/w. When formulated as an ointment, the active ingredient may be employed with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water emulsion base.

If desired, the aqueous phase of the cream base may include polyhydric alcohols, i.e., alcohols having two or more hydroxyl groups such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerol, and polyethylene glycols (including PEG 400), and mixtures thereof. Topical formulations may desirably include compounds that enhance absorption or penetration of the active ingredient through the skin or other relevant areas. Examples of such transdermal penetration enhancers include dimethyl sulfoxide and related analogs.

The oily phase of the emulsions of the invention may be constituted in a known manner by known ingredients, including mixtures of at least one emulsifier with fats or oils, or mixtures of at least one emulsifier with fats and oils. Preferably, a hydrophilic emulsifier is included with a lipophilic emulsifier as a stabilizer. The emulsifier together with/without the stabilizer constitutes an emulsifying wax, and the wax together with the oil and fat constitutes the emulsifying ointment base, which forms the oily dispersed phase of the cream formulation. Emulsifiers and emulsion stabilizers suitable for use in the formulation include60、80. Cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

Aqueous suspensions of pharmaceutical formulations contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as naturally occurring phosphatides (e.g. lecithin), condensation products of an alkylene oxide with fatty acids (e.g. polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g. heptadecaethylene oxide cetyl alcohol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g. polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl paraben), one or more coloring agents, one or more flavoring agents, and one or more sweetening agents (e.g., sucrose or saccharin).

The pharmaceutical compositions may be in the form of sterile injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions. Such suspensions may be formulated according to methods known in the art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. Sterile injectable preparations may also be solutions or suspensions in a non-toxic parenterally-acceptable diluent or solvent, for example, as solutions in 1, 3-butanediol, or as lyophilized powders. Among the acceptable carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may be conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may also be used in the preparation of injectables.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain about 1-1000mg of the active substance, complexed with a suitable and convenient amount of carrier material, which may vary from about 5 to about 95% of the total composition (weight: weight). The pharmaceutical composition can be prepared to provide an easily measured amount of administration. For example, an aqueous solution intended for intravenous infusion may contain about 3-500 μ g of active ingredient per mL of solution so that an appropriate volume of infusion at a rate of about 30mL/hr may be produced.

Formulations suitable for parenteral administration include aqueous and sterile anhydrous injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended subject; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active ingredient. The active ingredient is preferably present in the formulation at a concentration of about 0.5-20% w/w, such as about 0.5-10% w/w, about 1.5% w/w.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or nasal administration have a particle size, for example, in the range of 0.1 to 500 microns (including particle sizes intermediate the 0.1 and 500 micron range, with incremental microns being, for example, 0.5, 1, 30 microns, 35 microns, etc.), and are administered by rapid inhalation through the nasal cavity, or by inhalation into the oral cavity, so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents, for example, compounds heretofore used in the treatment or prevention of conditions described below.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The formulations may also be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier for injections, for example water, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, or a suitable fraction thereof, of an active ingredient as herein above recited.

The present invention also provides a veterinary composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and at least one chemotherapeutic agent as defined above, together with a veterinary carrier. Veterinary carriers are materials that can be used for the purpose of administering the composition and can be solid, liquid, or gaseous substances that are inert or acceptable in the veterinary art and that are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally, or by any other desired route.

Combination therapy

The compounds of formula I or pharmaceutically acceptable salts thereof may be used in combination with other chemotherapeutic agents for the treatment of hyperproliferative diseases or conditions, including tumors, cancers, and neoplastic tissues, as well as premalignant and non-neoplastic or non-malignant hyperproliferative diseases. In certain embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, is combined with a second compound having anti-hyperproliferative properties, or for use in the treatment of hyperproliferative diseases, in a dosing regimen that is a combination therapy. The second compound of the dosing regimen preferably has complementary activities to the compound of formula I or a pharmaceutically acceptable salt thereof, and such that they do not adversely affect each other. The compounds may be administered in an amount effective for the intended purpose. In one embodiment, the therapeutic combination is administered by a dosing regimen in which a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof is administered in a range from twice daily to once every three weeks (q3wk) and a therapeutically effective amount of a chemotherapeutic agent is administered in a range from twice daily to once every three weeks.

The combination therapy may be administered as a simultaneous or sequential dosing regimen. When administered first and later, the combination may be administered in two or more administrations. Combined administration includes co-administration using separate formulations, as well as sequential administration in any order, wherein preferably there is a period of time during which both (or all) active ingredients simultaneously exhibit their biological activity.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof can be administered for a period of about 1 to about 10 days after the start of administration of the one or more agents. In another particular aspect of the invention, the compound of formula I or a pharmaceutically acceptable salt thereof may be administered for a period of about 1 to about 10 days before the start of administration of the combination. In another particular aspect of the invention, the administration of the compound of formula I or a pharmaceutically acceptable salt thereof and the administration of the chemotherapeutic agent begin on the same day.

Suitable dosages for any of the above co-administered drugs are those currently used and may be reduced because of the combined effect (synergy) of the newly identified drug with other chemotherapeutic agents or treatments, such as increasing the therapeutic index or lessening toxicity or other side effects or consequences.

In particular embodiments of anti-cancer treatment, the compound of formula I or a pharmaceutically acceptable salt thereof may be combined with a chemotherapeutic agent, as well as with surgical treatment and radiation therapy. The amounts of the compound of formula I or a pharmaceutically acceptable salt thereof and the other pharmaceutically active chemotherapeutic agent, and the relative time periods of administration, will be selected to achieve the desired combined therapeutic effect.

Administration of pharmaceutical compositions

The compounds may be administered by any route appropriate to the condition being treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, inhalation, intradermal, intrathecal, epidural, and infusion techniques), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary, and intranasal administration. Topical administration may also involve transdermal administration such as the use of a transdermal patch or iontophoresis device.

Pharmaceutical preparations are described in Remington's Pharmaceutical Sciences,18^thEd., (1995) Mack Publishing co., Easton, PA. Other examples of Pharmaceutical formulations can be found in Liberman, h.a. and lachman, l., eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol 3,2^ndEd, New York, NY. For local immunosuppressive therapy, the compound may be administered intralesionally, including by perfusion or otherwise contacting the graft with the inhibitor prior to transplantation. It will be appreciated that the preferred route may vary with, for example, the condition of the recipient. When the compound is administered orally, it can be formulated into pills, capsules, tablets, and the like, with a pharmaceutically acceptable carrier, glidant, or excipient. When the compound is administered parenterally, it may be formulated with a pharmaceutically acceptable parenteral vehicle or diluent, and it is in unit dose injectable form as described in detail below.

The dose range for treating a human patient may be from about 20mg to about 1600mg daily of a compound of formula I, or a pharmaceutically acceptable salt thereof. A typical dose may be about 50mg to about 800mg of the compound. The dose may be administered once daily (QD), twice daily (BID), or more frequently, depending on the Pharmacokinetic (PK) and Pharmacodynamic (PD) properties of the particular compound, including absorption, distribution, metabolism, and excretion. In addition, toxicity factors can affect dosage and dosing regimens. When administered orally, the pill, capsule, or tablet may be ingested twice daily, once daily, or less frequently, such as once weekly or once biweekly or once every three weeks for a specified period of time. The dosing regimen may be repeated for numerous cycles of treatment.

Method of treatment

(1) A therapeutic combination of a compound of formula I, or a pharmaceutically acceptable salt thereof, and (2) a chemotherapeutic agent, is useful for treating diseases, conditions and/or disorders, including but not limited to those modulated by AKT kinase in mammals. Cancers that may be treated according to the methods of the present invention include, but are not limited to, mesothelioma, endometrial, glioma, pancreatic, breast, lung, ovarian, prostate, melanoma, gastric, colon, head and neck.

It has been determined that certain combinations of the invention provide improved effects against certain cancer phenotypes. For example, certain combinations of the invention provide improved efficacy against cancers associated with PTEN mutations (or low or null states), AKT mutations (or high pAKT expression or amplification levels), PI3K mutations, Her2/ErbB2 amplification, RAS mutations, RAF mutations, or combinations thereof.

Thus, certain combinations described herein may be particularly effective against these types of cancer.

For example, in colorectal cancer, a combination of PI3k/AKT mutations (e.g., PI3K H1047R, E545K, D549N, P421L, L568F, L569F, P449T, or a combination thereof) and RAS/RAF mutations (KRAS G13D, G12D, G12V, or a combination thereof) is expected to respond strongly to the present application combination and a strong synergy is observed for the combination of GDC-0068 and GDC-0973.

Also, in non-small cell lung cancer, a strong synergistic effect was observed for the combination of GDC-0068 and GDC-0973, where: (i) there is a combination of PI3k/AKT mutation (PI3k E545K, L997P, M772X, N996H, or a combination thereof) and RAS/RAF mutation (Q61H, G12C, Q61K, N85K, G12S, BRAF V600E, or a combination thereof), and (ii) there is a combination of RAS/RAF without PI3k mutation.

Also, in melanoma, a strong synergy was observed for the combination of GDC-0068 and GDC-0973, where: (i) there is a BRAF V600E mutation, and (ii) there is a BRAF V600E mutation or deletion or amplification with a PTEN mutation (null or low status) or with a high pAKT expression or activity level.

Kits for testing whether a patient has the BRAF V600E mutation are commercially available. One example is4800 BRAF V600 mutation test (Roche Molecular Systems Inc.), which detects BRAF V600E mutations in formaldehyde-fixed paraffin-embedded (FFPET) human melanoma tissue. It has been approved in the united states as a combination diagnostic for treatment with vemurafenib (vemurafenib) or a pharmaceutically acceptable salt thereof, designed to treat patients in which melanoma harbors a mutated form of the BRAF gene. In both pre-clinical and clinical studies,the BRAF mutation test is used for detecting BRAF V600E (1799T)>A) A positive identity of 97.3% was found in the mutation, indicating that>All BRAF mutations were reported in the cosinc database to 85%.

In formaldehyde-fixed paraffin-embedded tissue (FFPET),BRAF mutation test detectable>V600E mutation at 5% mutation level. The test can also detect other V600 mutations such as V600D and V600K.The BRAF mutation test may be performed from a received sample (such as obtained from a patient)Tissue sample or tumor cell of (2)<Within 8 hours.4800 BRAF V600 mutation was tested as4800 system, real-time PCR test on V2.0, and it is intended for use in the assisted selection of melanoma patients in which the tumor carries the BRAF V600E mutation.

PTEN null (or low) status may be measured by any suitable method known in the art. In one example, IHC is used. Alternatively, protein spotting can be used for analysis. Antibodies to PTEN are commercially available (Cell Signaling Technology, Beverly, MA, Cascade Biosciences, Winchester, MA). Exemplary procedures for IHC and protein spotting analysis for PTEN status are described in Neshat, M.S. et al, enhancement sensitivity of PTEN-specific analytes to inhibition of FRAP/mTOR, Proc. Natl Acad. Sci. USA 98, 10314-. In addition, cancers associated with AKT mutations, PI3K mutations, and Her2/ErbB2 amplification can be identified using techniques known in the art.

The level of activation or phosphorylation of AKT ("pAKT") in a given sample compared to the level of non-activated or non-phosphorylated AKT can be measured by methods known in the art. The pAKT status can be expressed in terms of a ratio (e.g., the amount of pAKT in a tumor cell divided by the amount of pAKT in a non-tumor cell of the same type) or a subtraction (e.g., the amount of pAKT in a tumor cell minus the amount of pAKT in a non-tumor cell of the same type). The pAKT profile can also be expressed as follows: the level of activation pathway obtained by measuring the amount of phosphorylated downstream targets (e.g. pGSK or PRAS40) of AKT. A high pAKT profile means that the activation or phosphorylation level of total AKT in the sample is above baseline. In one example, the baseline value is a basal level of pAKT for a given cell type. In other examples, the baseline value is the average or mean level of pAKT in a given sample cell population, e.g., non-cancerous cells. In other examples, high pAKT refers to tumor cells that overexpress or expand phosphorylated or activated AKT in the cells when compared to the average of normal, healthy (e.g., non-tumor) cells of the same type from the same mammal or patient population. The pAKT profile can also be used in combination with other markers (e.g. FOXO3a localization profile) to predict the efficacy of certain PI3k/AKT kinase pathway inhibitors, or for example with BRAF V600E mutation status to predict the efficacy of certain combinations of compounds of formula I with vemurafenib, particularly in patients with vemurafenib resistant cancers such as metastatic or unresectable melanoma. Kits for measuring pAKT in tissue samples are commercially available (e.g., phospho-Akt (Thr308) STAR ELISA kit, emdmillibore).

Kits for testing for the presence of PI3k, KRAS and AKT mutations are commercially available (Qiagen).

In a particular aspect, the invention provides a method for treating a patient having a cancer associated with PTEN mutation or loss of expression, AKT mutation or amplification, PI3K mutation or amplification, Her2/ErbB2 mutation or amplification, KRAS mutation or amplification, BRAF mutation or amplification or a combination thereof, comprising administering the combination to the patient. In another aspect, the invention provides a method for identifying a patient having a cancer treatable with a combination of the invention, comprising determining whether the patient's cancer is associated with PTEN mutation or loss of expression, AKT mutation or amplification, PI3K mutation or amplification or Her2/ErbB2 amplification, KRAS mutation or amplification, BRAF mutation or amplification or a combination thereof, wherein the association of the patient's cancer with PTEN mutation or loss of expression, AKT mutation or amplification, PI3K mutation or amplification or Her2/ErbB2 amplification, KRAS mutation or amplification, BRAF mutation or amplification or a combination thereof predicts that the cancer is treatable with a combination of the invention. In another aspect, the invention provides a method further comprising treating a patient so identified with a combination of the invention. In one embodiment, the cancer is ovarian cancer, breast cancer, melanoma, colon cancer, or non-small cell lung cancer.

Article of manufacture

In a further embodiment of the invention, there is provided an article of manufacture or "kit" containing a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of diseases and conditions as described above. In one embodiment, the kit comprises a container and a compound of formula I or a pharmaceutically acceptable salt thereof.

The kit may further comprise a label or package insert on or in association with the container. The term "package insert" refers to instructions typically included in commercial packaging for therapeutic products, including an introduction to such therapeutic products regarding indicators, uses, dosages, administrations, contraindications and/or warnings concerning use. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container may contain an amount of a compound of formula I or a pharmaceutically acceptable salt or formulation thereof effective to treat the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag that may be pierced with a hypodermic needle or a vial with a stopper). At least one active agent in the composition is a compound of formula I or a pharmaceutically acceptable salt thereof. The label or package insert indicates that the composition is for use in treating a selected condition, such as cancer. In one embodiment, the label or package insert indicates that compositions comprising a compound of formula I or a pharmaceutically acceptable salt thereof are useful for treating conditions resulting from abnormal cell growth. The label or package insert may also indicate that the composition may be used to treat other conditions. Alternatively or additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further comprise other desirable materials (from a commercial and user standpoint), including other buffers, diluents, filters, needles and syringes.

The kit may further comprise instructions for administering the compound of formula I or a pharmaceutically acceptable salt thereof and the second pharmaceutical formulation (if present). For example, if the kit comprises a first composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a second pharmaceutical formulation, the kit may further comprise instructions for administering the first and second pharmaceutical compositions to a patient in need thereof simultaneously, sequentially or separately.

In a further embodiment, the kit is suitable for delivering a compound of formula I or a pharmaceutically acceptable salt thereof in solid oral form, such as a tablet or capsule. Preferably, such kits comprise a plurality of unit doses. Such kits may comprise cards having the dosages oriented in their intended order of use. An example of such a kit is a "blister pack". Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. If desired, memory aids may be provided, for example in the form of numbers, letters or other indicia, or with a schedule indicating the number of days in the treatment schedule on which the dose may be administered.

According to one embodiment, a kit may comprise (a) a first container having a compound of formula I or a pharmaceutically acceptable salt thereof contained therein; and optionally (b) a second container having a second pharmaceutical formulation contained therein, wherein the second pharmaceutical formulation comprises a second compound having anti-hyperproliferative activity. Alternatively or additionally, the kit may further comprise a third container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further comprise other desirable materials (from a commercial and user standpoint), including other buffers, diluents, filters, needles and syringes.

When the kit comprises a composition of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a second therapeutic agent, i.e., a chemotherapeutic agent, the kit may comprise a container for holding the separate compositions, e.g., separate bottles or separate foil packages, however, the separate compositions may also be contained within a single, undivided container. Typically, the kit contains instructions for administering the individual components. The kit form is particularly advantageous when separate components administered in different dosage forms (e.g., oral and parenteral), administered at different dosage intervals, or when the prescribing physician desires to determine the dosage of the individual components of the combination.

Detailed aspects of the invention

In a particular aspect of the invention, the hyperproliferative disease is cancer.

In a particular aspect of the invention, the cancer is associated with a PTEN mutation.

In a particular aspect of the invention, the cancer is associated with AKT mutation, overexpression or amplification.

In a particular aspect of the invention, the cancer is associated with a PI3K mutation.

In a particular aspect of the invention, the cancer is associated with a KRAS mutation.

In a particular aspect of the invention, the cancer is associated with a BRAF mutation.

In a particular aspect of the invention, the cancer is associated with a combination of (1) a PTEN, AKT or PI3K mutation and (2) a KRAS or BRAF mutation. In one example, the cancer is ovarian cancer, breast cancer, melanoma, colon cancer, or non-small cell lung cancer.

In a particular aspect of the invention, the cancer is resistant to one or both of GDC-0068 and GDC-0973 single agent therapy, but responds to a combined treatment of GDC-0068 and GDC-0973. In one example, the cancer is ovarian cancer, breast cancer, melanoma, colon cancer, or non-small cell lung cancer.

In a particular aspect of the invention, the cancer is selected from mesothelioma, endometrial, pancreatic, breast, lung, ovarian, prostate (e.g. castration resistant prostate), melanoma, gastric, colon, renal, head and neck, and glioma.

In a particular aspect of the invention, the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered orally.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof and the cancer is pancreatic cancer.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or a pharmaceutically acceptable salt thereof and the cancer is pancreatic cancer.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof and the cancer is colon cancer.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof and the cancer is breast cancer.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof and the cancer is ovarian cancer.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof and the cancer is lung cancer.

In a particular aspect of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is administered in combination with GDC-0973 or PD-0325901 or a pharmaceutically acceptable salt thereof and the cancer is melanoma.

In one particular aspect of the invention, the compound of formula I or a pharmaceutically acceptable salt thereof is formulated as a tablet.

Examples

For the purpose of illustrating the invention, the invention includes the following embodiments. It should be understood, however, that these examples are not limiting and are intended only to set forth ways of practicing the invention.

Example 1

(S) -2- (4-chlorophenyl) -1- (4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta[d]Pyrimidin-4-yl) piperazin-1-yl) -3- (isopropylamino) propan-1-one

Step 1Using dry ice-isopropylThe propanol bath a solution of ethyl pregelatonate (130g,662 mmol) in EtOAc (900mL) was cooled to-78 ℃. The mixture was allowed to ozonolysis until the reaction mixture turned purple. At this point, ozone generation ceased and the reaction mixture was removed from the dry ice bath. Oxygen was bubbled through the reaction mixture until it turned yellow. The reaction mixture was concentrated in vacuo, and the resulting residue was dissolved in glacial acetic acid (400 mL). The solution was cooled to 0 ℃ and zinc dust (65g,993mmol) was added portionwise over 30 minutes. The reaction mixture was then stirred for 2 hours, at which time the reaction mixture was filtered through a plug of celite to remove the zinc fines. The acetic acid is treated with aqueous NaOH and NaHCO₃Neutralized to pH 7 and extracted with diethyl ether (3X 800 mL). The combined organics were washed with brine, MgSO₄Drying and concentration gave (2R) -2-methyl-5-oxocyclopentane-carboxylic acid ethyl ester as a brown liquid (107g, 95%).

Step 2Ammonium acetate (240.03g,3113.9mmol) was added to a solution of ethyl (R) -2-methyl-5-oxocyclopentanecarboxylate (106.0g,622.78mmol) in MeOH (1.2L). The reaction mixture was stirred at room temperature under nitrogen for 20 hours before it was judged to be complete by TLC and HPLC. The reaction mixture was concentrated and MeOH was removed. The resulting residue was dissolved in DCM and washed with H₂O twice, once with brine, and dried (Na)₂SO₄) Filtration and concentration gave ethyl (R) -2-amino-5-methylcyclopent-1-enecarboxylate (102g, 97% yield) as an orange oil. LC/MS (APCI +) M/z 170 [ M + H ]]+.

Step 3A solution containing ethyl (R) -2-amino-5-methylcyclopent-1-enecarboxylate (161.61g,955.024 mmol) and ammonium formate (90.3298g,1432.54mmol) in formamide (303.456ml,7640.19 mmol) was heated to an internal temperature of 150 ℃ and stirred for 17 hours. The reaction mixture was cooled and transferred to a 2L single-necked flask. The excess formamidine was then removed by high vacuum distillation. Once formamidine stopped being produced, the remaining oil in the still pot was dissolved in DCM and washed with brine (3X 200 mL). The combined aqueous washes were extracted with DCM. The combined organic extracts were dried (Na)₂SO₄) Filtered and concentrated. The resulting brown oil is dissolved to a minimumIn DCM, and this solution was added to a stirred solution of ether (ca. 5vol ether/DCM solution) using a separatory funnel, which caused some brown precipitate to form. The brown precipitate was removed by filtration through a medium glass frit funnel, rinsed with ether and worked up. Concentrating the filtrate, grinding with diethyl ether for more than two times, and drying in high vacuum line to obtain (R) -5-methyl-6, 7-dihydro-5H-cyclopenta [ d]Pyrimidin-4-ol (93.225g, 65.00% yield) as a tan paste solid. LC/MS (APCI-) m/z 149.2.

Step 4Pure POCl₃(463.9ml,5067mmol) was added slowly via the addition funnel to (R) -5-methyl-6, 7-dihydro-5H-cyclopenta [ d ]]Solution of pyrimidin-4-ol (152.2g,1013mmol) in DCE (1.2L) at 0 ℃. After the addition was complete, the reaction mixture was warmed to room temperature, then heated to reflux and stirred for 70 minutes. The reaction was complete as determined by HPLC. The reaction mixture was cooled to room temperature and the excess of POCl was added₃Quench in 4 parts as follows: the reaction mixture was transferred to a separatory funnel and dropped to a container containing ice and saturated NaHCO cooled in an ice bath₃Beaker of solution. Once the addition of the reaction mixture to each portion was complete, the quenched mixture was stirred for 30 minutes to ensure POCl before transfer to a separatory funnel₃Is completely destroyed. The mixture was transferred to a separatory funnel and extracted twice with DCM. The combined extracts were dried (Na)₂SO₄) Filtered and concentrated. The crude material was purified on silica gel as follows: silica gel (1kg) was slurried in 9:1 hexane: ethyl acetate in a 3L frit funnel, the silica gel was allowed to stand under vacuum and capped with sand. The crude material was loaded with a DCM/hexane mixture and the compound was eluted under vacuum using a 1L manifold flask. The high Rf by-product is eluted first followed by (R) -4-chloro-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ]]Pyrimidine (104.4g, 61.09% yield) as a brown oil. Triethylamine (93.0ml,534mmol) and tert-butyl piperazine-1-carboxylate (34.8g,187mmol) were added to (R) -4-chloro-5-methyl-6, 7-dihydro-5H-cyclopenta [ d [ -d]Pyrimidine (30.0g,178mmol) in n-BuOH (250 mL). The reaction mixture was heated to reflux under nitrogen and stirred overnight (17 hours) before it was concentrated on Rotavap. Will be provided withThe resulting oil was dissolved in DCM and washed with H₂O washing and drying (Na)₂SO₄) Filtered and concentrated. The resulting brown oil was purified on silica gel (first eluting with 2:1 hexanes: ethyl acetate until the product was cleanly eluted, then eluting with a gradient of 1:1-1:5 DCM: ethyl acetate) to afford (R) -4- (5-methyl-6, 7-dihydro-5H-cyclopenta [ d ]]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (42.0g, 74.1% yield) as a beige powder. LC/MS (APCI +) M/z 319.1[ M + H ]]⁺.

Step 577% solids max. MCPBA (23.9g,107mmol) was added portionwise to (R) -4- (5-methyl-6, 7-dihydro-5H-cyclopenta [ d%]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (20.0 g,62.8mmol) in CHCl₃(310mL) at 0 ℃. The reaction mixture was stirred for 5 minutes, then warmed to room temperature and stirred for 90 minutes. Similar HPLC was observed after 7.5 hours. The reaction mixture was cooled to 0 ℃ and then NaHCO was added₃(13.2g,157mmol) and a further 0.5 equivalent of m-CPBA. The reaction mixture was stirred overnight (14 hours). The reaction mixture was cooled to 0 ℃ and Na was added dropwise via a dropping funnel₂S₂O₃(29.8g,188mmol) in H₂Solution in O (50 mL). Then add Na via a dropping funnel₂CO₃(24.6g,232mmol) in H₂Solution in O (70mL) (mixture becomes homogeneous). The reaction mixture was stirred for 30 minutes, then the mixture was taken up in CHCl₃(3X 150 mL). The combined extracts were dried (Na)₂SO₄) Filtered and concentrated to obtain the N-oxide. LC/MS (APCI +) M/z 335.1[ M + H ]]+.

Step 6A reaction of Ac₂O (77.0ml,816mmol) was added to the N-oxide from step 5 (21.0g, 62.8 mmol). The reaction mixture was heated in a 90 ℃ sand bath under nitrogen and stirred for 100 minutes. The reaction mixture was cooled to room temperature and excess acetic anhydride was removed by rotary evaporation. The resulting oil was dissolved in DCM, which was then carefully poured over ice saturated Na₂CO₃In (1). The mixture was extracted with DCM and the combined extracts were dried (Na)₂SO₄) Filtered and concentrated to obtain (5R) -4- (7-acetoxyl-5-methyl-6, 7-dihydro-5H-cyclopenta [ d]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (23.6g, 100%) as a brown foam. LC/MS (APCI +) M/z 377.1[ M + H ]]+.

Step 7Reacting LiOH-H₂O (6.577g,156.7mmol) was added to (5R) -4- (7-acetoxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ]]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (23.6g,62.69 mmol) in 2:1 THF H₂O (320mL) in a 0 ℃ solution. The reaction mixture was stirred for 10 minutes and then warmed to room temperature. The same LC/MS was observed at 3 hours and 4.5 hours. The reaction mixture was cooled to 0 ℃ and then saturated NH was added₄Cl was added to the mixture. The mixture was stirred for 5 minutes and most of the THF was removed by rotary evaporation. The mixture was extracted with EtOAc (3X 250mL) and the combined extracts were dried (Na)₂SO₄) Filtered and concentrated. The crude material was purified by flash chromatography on a Biotage 65M (4:1 DCM: ethyl acetate followed by a gradient of 1:1 to 1:4 DCM: ethyl acetate). Once the product eluted, the column was washed with ethyl acetate. The remaining product (8.83g) was then eluted with 30:1 DCM: MeOH. Purification of fractions of the mixture by flash chromatography using Biotage 40M under the same conditions again gave an additional 2.99g which gave a combined yield of (5R) -4- (7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d [ d ] ])]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (11.82g, 56.38% yield) as a brown foam. LC/MS (APCI +) M/z 335.1[ M + H ]]+.

Step 8A solution of DMSO (5.45mL,76.8mmol) in DCM (50mL) was added dropwise via the addition funnel to a-78 deg.C solution of oxalyl chloride (3.35mL,38.4mmol) in DCM (150 mL). The reaction mixture was stirred for 35 minutes, then (5R) -4- (7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ] was slowly added via a dropping funnel]A solution of t-butyl pyrimidin-4-yl) piperazine-1-carboxylate (9.17g,27.4 mmol) in DCM (80 mL). The reaction mixture was stirred at-78 ℃ for a further 1h, after which pure triethylamine (18.0ml,129mmol) was added to the mixture. The reaction mixture was then warmed to room temperature and then stirred for 30 minutes. Addition of H₂And O. The mixture was extracted with DCM (3 × 200 mL) and the combined extracts were dried (Na)₂SO₄) Filtered and concentrated in vacuo. The crude material was purified on silica gel (Biotage 65M) (the column was washed with about 800mL 4:1 DCM: EtOAc, then a gradient of 1:1 DCM: ethyl acetate until the product eluted, then the product eluted with 1:4 DCM: EtOAc) to give (R) -4- (5-methyl-7-oxo-6, 7-dihydro-5H-cyclopenta [ d)]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (7.5g, 82.3% yield) as a brown foam. The foam was concentrated from DCM/hexane (3 ×) to give a very light brown foam. HPLC>95% area. LC/MS (APCI +) M/z 333[ M + H ]]+.

Step 9Triethylamine (4.33ml,31.1 mmol; degassed with nitrogen for 30 minutes before use) and formic acid (1.36ml,36.1 mmol; degassed with nitrogen for 30 minutes before use) were added to (R) -4- (5-methyl-7-oxo-6, 7-dihydro-5H-cyclopenta [ d [ -d ] - ])]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (9.75g, 29.3mmol) in DCM (210 mL; degassed with nitrogen for 30 minutes before use). The mixture was stirred for 5 minutes, then Ru catalyst (0.0933g,0.147mmol) was added. The reaction mixture was stirred under a positive pressure of nitrogen overnight (18 hours). The reaction mixture was concentrated to dryness and dried under high vacuum. The impure material was purified by flash chromatography on Biotage 65M (loaded with 1:1 DCM: ethyl acetate 500mL, washed, then 1:4 DCM: ethyl acetate until the product eluted (second spot), then the gradient was pure ethyl acetate, then the remaining product was eluted with 25:1 DCM: MeOH). The fractions were combined and concentrated on a rotary evaporator. The residue was again concentrated with DCM/hexane to give 4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d [)]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (predominantly) and 4- ((5R,7S) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d [ -d [ ]]A mixture of t-butyl pyrimidin-4-yl) piperazine-1-carboxylate (minor) (9.35g, 95.3% yield) as a beige foam. LC/MS (APCI +) M/z 335[ M + H +]+. 1H NMR (CDCl3) showed 88% de by integration of the carbinol methine.

Step 104-nitrobenzoyl chloride (4.27g,23.0mmol) was added to 4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ]]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (7.0g,20.9 mmol) and triethylamine (4.38mL,31.4mmol) in DCM (110mL)At 0 deg.C. The reaction mixture was stirred at room temperature overnight, after which saturated NaHCO was added₃. The mixture was stirred for 10 min and then extracted with DCM. The combined extracts were dried (Na)₂SO₄) Filtered and concentrated. The crude material was purified by flash chromatography on a Biotage 65M column (3: 1 hexanes: ethyl acetate to load the product, then 2:1 hexanes: ethyl acetate to elute 4- ((5R,7R) -5-methyl-7- (4-nitrobenzoyloxy) -6, 7-dihydro-5H-cyclopenta [ d ] c]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester and a small amount of mixed fractions). The 4- ((5R,7S) -5-methyl-7- (4-nitrobenzoyloxy) -6, 7-dihydro-5H-cyclopenta [ d ] was then eluted using 1:2 hexanes: ethyl acetate]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester. Concentrating the product-containing fraction by rotary evaporation to obtain 4- ((5R,7R) -5-methyl-7- (4-nitrobenzoyloxy) -6, 7-dihydro-5H-cyclopenta [ d]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (8.55g, 84.5% yield) as a yellow foam. LC/MS (APCI +) M/z 484[ M + H +]+. 1H NMR (CDCl3) showed a single diastereomer). Concentrating the fraction containing other diastereoisomers by rotary evaporation to obtain 4- ((5R,7S) -5-methyl-7- (4-nitrobenzoyloxy) -6, 7-dihydro-5H-cyclopenta [ d]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (0.356g, 3.52% yield) as a brown foam. LC/MS (APCI +) M/z 484[ M + H +]+.

Step 11Addition of LiOH-H2O (0.499g,11.9mmol) to 4- ((5R,7R) -5-methyl-7- (4-nitrobenzoyloxy) -6, 7-dihydro-5H-cyclopenta [ d]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (2.30g,4.76mmol) in 2:1 THF H₂O (40mL) at 0 ℃. The reaction mixture was warmed to room temperature and stirred for 1 hour. THF was removed by rotary evaporation and saturated NaHCO was added₃And the mixture was extracted with ethyl acetate. The combined extracts were extracted with saturated NaHCO₃Washed (1X) and dried (Na)₂SO₄) Filtering, and concentrating to obtain 4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d)]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (1.59g, 100.0% yield) as a yellow foam. HPLC generation after work-up>98 area% purity. LC/MS (APCI +) M/z 335[ M + H +]+。Preparation of 4- ((5R,7S) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ] using a similar procedure]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester.

Step 12Adding 4M HCl/dioxane (11.2ml,44.9mmol) to 4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d [)]Pyrimidin-4-yl) piperazine-1-carboxylic acid tert-butyl ester (0.600g, 1.79mmol) in dioxane (15 mL). The reaction mixture was stirred at room temperature overnight (20 hours) under nitrogen. The mixture was concentrated to dryness and dried on a high vacuum line. The crude material was suspended in ether, sonicated, and stirred for 5 minutes. The solid was isolated by filtration through a medium glass frit funnel under nitrogen pressure, rinsed with diethyl ether, dried under nitrogen pressure and further dried on a high vacuum line to give (5R,7R) -5-methyl-4- (piperazin-1-yl) -6, 7-dihydro-5H-cyclopenta [ d [ -d]Pyrimidin-7-ol dihydrochloride (0.440g, 79.8% yield) as a yellow powder. LC/MS (APCI +) m/z 235. Preparation of (5R,7S) -5-methyl-4- (piperazin-1-yl) -6, 7-dihydro-5H-cyclopenta [ d ] using an analogous procedure]Pyrimidin-7-ol dihydrochloride.

Step 13Methyl 2- (4-chlorophenyl) acetate (36.7g,199mmol) and paraformaldehyde (6.27g, 209mmol) were dissolved/suspended in DMSO (400mL) and treated with NaOMe (537mg,9.94 mmol). The mixture was stirred at room temperature for 2 hours and complete by TLC analysis of the crude material. The reaction mixture was poured into ice-cold water (700 mL; white emulsion) and neutralized by addition of 1M HCl solution. The aqueous layer was extracted with ethyl acetate (3 ×), and the organic layers were combined. The organic layer was washed with water (2X), washed with brine (1X), separated, over MgSO₄Drying, filtration, and concentration in vacuo afforded the crude product as a yellow oil. The residue was loaded onto a large frit filter containing silica gel and eluted with 9:1 hexanes: ethyl acetate until starting material/olefin collection. The packing was then eluted with 1:1 hexane: ethyl acetate until the pure desired product was completely eluted. The pure fraction was concentrated to give methyl 2- (4-chlorophenyl) -3-hydroxypropionate as a colorless oil (39.4g, 92%).

Step 14Methyl 2- (4-chlorophenyl) -3-hydroxypropionate (39.4g,184mmol) was dissolved in DCM (500mL) and TEA (64.0mL,459mmol) was usedAnd (6) processing. The solution was cooled to 0 ℃ and treated slowly with MsCl (15.6mL,202mmol), then stirred for 30 min, and was shown to be complete by TLC analysis. The solution was partitioned with 1N HCl solution and the aqueous layer was extracted once with DCM. The combined organic layers were washed more than once with 1N HCl solution, separated and diluted NaHCO₃The solution was washed and separated. The organic layer was purified over MgSO₄Dried, filtered, and concentrated in vacuo to give an orange oil. The residue was loaded onto a large frit filter with silica gel packing and eluted with 9:1 hexanes: ethyl acetate, which by TLC analysis showed the pure desired product. The pure fraction was concentrated to give methyl 2- (4-chlorophenyl) acrylate as a colorless oil (30.8g, 85%). The solution of methyl 2- (4-chlorophenyl) acrylate (500mg, 2.54mmol) in THF (1.35mL) was added to i-PrNH at 0 deg.C₂(217uL,2.54mmol) in THF (5.0 mL). The reaction mixture was stirred at room temperature overnight and analyzed by LCMS to show completion. Boc2O (584uL,2.54mmol) was added to the pipetted amine. The reaction mixture was stirred overnight and indicated completion by LCMS and TLC analysis of the mixture. The solution was concentrated in vacuo to give methyl 3- (tert-butoxycarbonyl (isopropyl) amino) -2- (4-chlorophenyl) propionate as a colorless oil (854mg, 94%). LC/MS (APCI +) M/z 256.1[ M-Boc ]]+.

Step 15Methyl 3- (tert-butoxycarbonyl (isopropyl) amino) -2- (4-chlorophenyl) propanoate (133g, 374mmol) was dissolved in THF (1.0L) and treated with KOTMS (56.0g,392mmol) at room temperature. The mixture was stirred overnight and LCMS analysis of the crude material showed completion. The mixture was concentrated in vacuo to give a wet foam which was dried under vacuum overnight to give potassium 3- (tert-butoxycarbonyl (isopropyl) amino) -2- (4-chlorophenyl) propionate as a white solid (148.7g, 105%). LC/MS (APCI +) M/z 242.1[ M-Boc-K]+.

Step 16:potassium 3- (tert-butoxycarbonyl (isopropyl) amino) -2- (4-chlorophenyl) propionate (77.2g, 203mmol) was dissolved in THF (515mL) and treated with pivaloyl chloride (26.3mL,213mmol) at room temperature. The mixture was stirred for 3 hours to form a mixed anhydride. (S) -4-benzyl oxazolidin-2-one (46.1g,260 mmol) was dissolved in THF (600mL) and cooled to-78 ℃ in a separate flask. The solution is treated with n-BuLi (102mL of a 2.50M solution in hexane, 254mmol) and stirred for one hour. The prepared anhydride solution was added via cannula to the stirred Li-oxazolidinone and the mixture was allowed to warm to room temperature overnight. The mixture was quenched by addition of saturated ammonium chloride solution and then partitioned between more water and ethyl acetate. The aqueous layer was extracted several times and the organic layers were combined. The organic layer was washed with water, then brine, separated and over MgSO₄Dried, filtered, and concentrated in vacuo. The residue was purified/separated (diastereomer) by chromatography (silica gel, eluting with 4:1 hexane: ethyl acetate) to give the fully separated diastereomer as a viscous oil: tert-butyl (R) -3- ((S) -4-benzyl-2-oxooxazolidin-3-yl) -2- (4-chlorophenyl) -3-oxopropyl (isopropyl) carbamate (12.16g, 24% acid racemate based on 1/2) and tert-butyl (S) -3- ((S) -4-benzyl-2-oxooxazolidin-3-yl) -2- (4-chlorophenyl) -3-oxopropyl (isopropyl) carbamate (39.14g, 77% acid racemate based on 1/2). LC/MS (APCI +) M/z 401.2[ M-Boc ]]+.

Step 17Reacting LiOH-H at room temperature₂O (168mg,4.00mmol) was added to a stirred solution of THF (30mL) and water (15mL) until it dissolved. The mixture was treated with hydrogen peroxide (658uL of a 35% wt. aqueous solution, 8.00mmol) and stirred at room temperature for 10 min. The reaction mixture was cooled to 0 ℃ with an ice bath and a solution of tert-butyl (S) -3- ((S) -4-benzyl-2-oxooxazolidin-3-yl) -2- (4-chlorophenyl) -3-oxopropyl (isopropyl) carbamate (1.00g,2.00mmol) in THF (15mL) was added dropwise over 10 minutes via a dropping funnel. The mixture was stirred at room temperature overnight and LCMS analysis of the crude material showed completion. The reaction mixture was cooled to 0 ℃ and then added over 10 minutes over 1M Na via a dropping funnel₂SO₃(9.00mL) solution treatment. After the addition was complete, the mixture was warmed to room temperature for 10 minutes. The mixture was concentrated, THF removed, and then diluted with water. The aqueous layer was washed twice with ethyl acetate (discarded). The aqueous layer was partitioned with ethyl acetate and then treated dropwise with 1M HCl with stirring until pH 2-3 was reached. The aqueous layer was extracted twice with ethyl acetate, and the organic layers were combined. The organic layer was washed with brine, separated and MgSO₄Dried, filtered, and concentrated in vacuo. The colorless oily product was dried under high vacuum for 1 hour to give (S) -3- (tert-butoxycarbonyl (isopropyl) amino) -2- (4-chlorophenyl) propionic acid as a viscous oil/foam (685mg, 100%). LC/MS (APCI +) M/z 242.1[ M-Boc +]+.

Step 18The (5R,7R) -5-methyl-4- (piperazine-1-yl) -6, 7-dihydro-5H-cyclopenta [ d]A solution of pyrimidin-7-ol dihydrochloride (2.92g,9.51mmol) and (S) -3- (tert-butoxycarbonyl (isopropyl) amino) -2- (4-chlorophenyl) propanoic acid (3.25g,9.51mmol) in DCM (40mL) and DIEA (5.0mL, 28.7mmol) was stirred at room temperature for 10 min. HBTU (3.61g,9.51mmol) was added to the mixture. The mixture was stirred at room temperature for 1 hour. The solvent was removed and the residue was dissolved in ethyl acetate (500mL) and washed with water (6X 100 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography (eluting with EtOAc-DCM/MeOH (20: 1)) to give (S) -2- (4-chlorophenyl) -3- (4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d ]]Pyrimidin-4-yl) piperazin-1-yl) -3-oxopropyl (isopropyl) carbamic acid tert-butyl ester (3.68g, 69%). LC/MS (APCI +) M/z 558.2[ M + H ]]+.

Step 19Mixing (S) -2- (4-chlorophenyl) -3- (4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d)]Pyrimidin-4-yl) piperazin-1-yl) -3-oxopropyl (isopropyl) carbamic acid tert-butyl ester (2.50g,4.48mmol) was dissolved in dioxane (22.4mL) and treated with a 4M HCl solution in dioxane (22.4mL,89.6mmol) at room temperature. The resulting solution was stirred overnight and LCMS analysis of the crude material showed completion. The solution was concentrated in vacuo to give a gum which was dissolved in a minimum amount of methanol (10 mL). The solution was pipetted into stirred ether (300mL) to give a white precipitate of the expected product. When the addition proceeded to about half, the white precipitate melted to a yellow gum. The material was concentrated in vacuo to a yellow gum which was allowed to stand overnight under reduced pressure to give (S) -2- (4-chlorophenyl) -1- (4- ((5R,7R) -7-hydroxy-5-methyl-6, 7-dihydro-5H-cyclopenta [ d [ -d [ ]]Pyrimidin-4-yl) piperazin-1-yl) -3- (isopropylamino) propan-1-one dihydrochloride as a pale yellow powder (2.14g, 90%).¹H NMR(D₂O,400MHz 8.39(s,1H),7.37-7.35(d,J＝8.4Hz,2H), 7.23-7.20(d,J＝8.4Hz,2H),5.29-5.25(m,1H),4.33-4.29(m,1H),4.14-4.10(m, 1H),3.89-3.19(m,11H),2.23-2.17(m,1H),2.08-1.99(m,1H),1.20-1.18(m, 6H),0.98-0.96(d,J＝6.8Hz,3H).MS(APCI+)[M+H]⁺458。

Example 2 in vitro cell proliferation assay

The in vitro potency of the combination of a compound of formula I with certain specific chemotherapeutic agents may be usedFluorescent cell survival assay (commercially available from Promega corp., Madison, WI) was measured. The homogenization assay (homogeneous assay) method is based on recombinant expression of homoptera luciferase (US 5583024; US 5674713; US5700670) and on the quantification of ATP present (an indicator of metabolically active cells) to determine the number of viable cells in culture (Crouch et al (1993) J. Immunol. meth.160: 81-88; US 6602677). The above-mentionedAssays were performed in 96 or 384 well format, making them amenable to automated High Throughput Screening (HTS) (Cree et al (1995) AntiCancer Drugs 6: 398-404). The homogeneous assay procedure comprises a single reagent(s) (ii)Reagent) was added directly to cells incubated in serum supplemented medium. There is no need for washing the cells, removing the medium and multiple pipetting steps. Within 10 minutes after adding the reagents and mixing, as few as 15 cells/well were detected systematically in 384-well format.

The homogeneous "add-mix-measure" mode results in cell lysis and produces a fluorescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The above-mentionedThe assay produces a "glow-type" fluorescent signal (produced by the luciferase reaction) which has a half-life typically greater than five hours, depending on the cell type and the type of cell being usedThe culture medium used. Viable cells are reflected in Relative Luminescence Units (RLU). The substrate, Beetle Luciferin (Beetle luciferase), is oxidatively decarboxylated by recombinant firefly luciferase, with conversion of ATP to AMP and production of photons. The extended half-life eliminates the need for reagent injectors and allows flexibility in processing in multi-plate continuous or multi-plate batch mode. The cell proliferation assay can be used in various multiwell formats, such as 96 or 384 well formats. Data may be recorded by a photometer or a CCD camera imaging device. The luminous output is presented, which is the Relative Light Units (RLU) measured over time.

Example 3 in vivo tumor xenograft potency

The efficacy of a representative combination of the invention is measured in vivo by implanting an allograft or xenograft of cancer cells in a rodent and treating the tumor bearing animal with the combination. Variable results may be expected depending on the cell line, the presence or absence of certain mutations in the tumor cells, the order of administration of the compounds, the dosing regimen, and other factors. Test mice were treated with drug or control (vehicle) and monitored over several weeks or longer to measure time to tumor volume doubling, log cell death value, and tumor inhibition. The results for representative combinations of the invention tested in this model are shown in the figure. The data in the figures show that the representative combinations provide improved results compared to the individual drugs administered alone.

Example 4 measuring PTEN status

PTEN status may be measured by any suitable method known in the art. In one example, IHC is used. Alternatively, protein spotting can be used for analysis. Antibodies to PTEN are commercially available (Cell signaling technology, Beverly, MA, Cascade Biosciences, Winchester, MA). Exemplary procedures for IHC and protein spotting analysis for PTEN status are described in Neshat, M.S. et al, enhanced sensitivity of PTEN-specific analytes to inhibition of FRAP/mTOR, Proc.Natl Acad.Sci.USA 98, 10314-. In addition, cancers associated with AKT mutations, PI3K mutations, and Her2/ErbB2 amplification can be identified using techniques known in the art.

Example 5 cell survival assay

Cells were seeded at a density of 1500 cells/well in a black, clear-bottomed 384-well plate (Catalog 353962; Becton Dickinson; Franklin Lakes, N.J.) and 5% CO at 37 deg.C₂Incubate overnight to 1.5 days. Serial dilutions of GDC-0068, GDC-0973, or a combination thereof were then added to the cells and incubated for an additional 96 hours. Cell viability was determined by measuring intracellular Adenosine Triphosphate (ATP) levels as described in the manufacturer's instructions (CellTiter-Glo fluorescent cell survival assay kit; Catalog G7573; Promega, Madison, Wis.). Fluorescence signals were recorded on an EnVision 2101 multi-tag reader (PerkinElmer; Waltham, MA).

Percent inhibition was calculated as follows: the quotient of 1 minus the Relative Light Units (RLU) for cells exposed to the combination of GDC-0068 and GCD-0973 divided by the RLU for cells exposed to DMSO is shown below:

% inhibition of 1- (RLU)_{Combination of}/RLU_DMSO)

BLISS analysis on expected% inhibition (E ═ E)_GDC-0068+E_GDC-0973-E_GDC-0068×E_GDC-0973) % inhibition by Experimental observations E_OBSA comparison is made. BLISS score as% inhibition E expected versus% inhibition E observed experimentally_OBSDifference (Δ E ═ E) of_OBS-E)。

The BLISS score quantifies the degree of potentiation from a single drug and a positive BLISS score indicates greater than simple additive effects. An overall BLISS score greater than 250 is considered a strong synergy observed over the range of concentrations tested.

An example of combined efficacy is shown as a heat map of three cancer cell lines: a2058, PTEN deficient and B-RAF^V600EA mutated melanoma cell line (see fig. 9); HCT-116 with PIK3CA^H1047RAnd KRAS^G13DA mutant colorectal cancer (CRC) cell line (see fig. 5); and NCI-H2122, havingWith KRAS^G12CA mutant non-small cell lung cancer (NSCLC) cell line (see fig. 7). A strong synergy with BLISS scores of ≧ 15 was observed for the individual doses at GDC-0068 concentrations between 0.37 and 10 μ M and GDC-0973 concentrations between 0.062 and 0.56 μ M in all three cell lines.

To further investigate whether the synergy between GDC-0068 and GDC-0973 is dependent on activation of the RAS/RAF and/or PI3K/Akt pathways, the combined effect was compared in a series of cell lines derived from melanoma patients: MALME3M B-RAF^V600EMetastatic melanoma cell lines and patient-matched MALME3 normal skin fibroblasts. MALME3M cells showed sensitivity to low concentrations of GDC-0973, and strong synergy was also observed at low concentrations of GDC-0973 and a wide range of concentrations of GDC-0068, despite the lack of single drug activity of GDC-0068 (see FIG. 28). In contrast, MALME3 cells were resistant to GDC-0973 and no synergy was observed in combination with GDC-0068 (see FIG. 29). Similarly, NCI-BL2122, which is a normal B lymphoblast cell derived from the same patient as the NSCLC cell line NCI-H2122, also did not show a synergistic response to the combination of GDC-0973 and GDC-0068 (see fig. 30), as opposed to the strong synergistic effect observed in NCI-H2122 cells (see fig. 7). These results indicate that the therapeutic benefit of the combination of MEK and Akt inhibitors can be selectively observed in cancer cells in which either the RAS/RAF pathway or both PI3K/Akt and RAS/RAF pathways are activated.

Example 6 protein Spot assay

In a petri dish (10 cm)²) Two million cells were seeded in a volume of 10mL and then at 37 ℃ in 5% CO₂Incubate overnight (about 16 hours). Cells were exposed to 1 and 3. mu.M GDC-0068, 0.25 and 0.75. mu.M GDC-0973 or 1. mu.M GDC-0068+ 0.25. mu.M GDC-0973 for 3 hours. After exposure, cells were washed with cold Phosphate Buffered Saline (PBS) and lysed in 1 × cell extraction buffer from Biosource (Carlsbad, CA) supplemented with protease inhibitors (Roche, Germany), 1mM phenylmethanesulfonyl fluoride (PMSF), and phosphatase inhibitor cocktail 1 and 2 from Sigma (st. Using the Bradford method (Bio-Rad protein assay (Bio-Rad; Hercules, C)A) The protein concentration is determined. For immunoblotting, identical protein amounts were separated by electrophoresis on a 4-20% triglycine gradient gel (Invitrogen; Carlsbad, Calif.); proteins were transferred to nitrocellulose membranes using the Criterion system and protocol from Bio-Rad.

Unless otherwise noted, the following antibodies, all from Cell Signaling Technologies (Beverly, MA), were used:

anti-pAkt (S473)

anti-pAkt (T308)

anti-pMEK 1/2(S217/221)

anti-pFoxo 1(T24)/FoxO3a (T32)

anti-pPRAS 40(T246)

Anti-p 4EBP1(T37/46)

anti-pERK 1/2(T202/Y204)

anti-pTSC 2(T1462)

anti-pS 6(S235/236)

anti-pS 6(S240/244)

Poly (ADP-ribose) polymerase (PARP) and cleaved PARP

GAPDH (from Advanced Immunochemical; Long Beach, CA)

To investigate the effect of the combination on Akt and MEK signaling, downstream targets of Akt and MEK were evaluated by protein spotting in HCT-116CRC cells exposed to 1 and 3 μ M GDC-0068, 0.25 and 0.75 μ M GDC-0973, or 1 μ M GDC-0068 in combination with 0.25 μ M GDC-0973, where synergy was observed. As shown in figure 24, a knock-down effect of the combination of downstream targets of Akt and MEK was observed at the combination, as well as an enhanced knock-down effect of several targets such as pTSC2, pS6(S235/236 and S240/244), PARP and cleaved PARP, which showed a better knock-down effect compared to the single drug used alone even at higher doses.

Example 7 flow cytometry assay

HCT-116 cells were seeded into 96-well tissue culture plates. At 37 deg.C, 5% CO₂After overnight incubation, cells were exposed to increasing concentrations of either GDC-0068 or GDC-0973 or a combination for 4 days. To detect apoptosis, 100. mu.L of cell suspension was added to a solution containing 4mM CaCl₂5 μ L of the dockerin V-Fluorescein Isothiocyanate (FITC) (BD Pharmingen; Franklin Lakes, N.J.) and 5 μ g/mL Propidium Iodide (PI) in 100 μ L PBS. The mixture was incubated on ice for 30 minutes and the cells were analyzed with a flow cytometer (BD Biosciences; San Jose, Calif.).

The percentage of propidium iodide- (PI) or dockerin V- (AV) positive cells was measured in each single drug or combination pair of GDC-0068 and GDC-0973, and the synergistic effects of cell death induction were analyzed by BLISS analysis. The combination results in PI compared to each single drug used alone⁺/AV⁺The percentage of cells increased and a strong synergy was observed between 0.37-10. mu.M GDC-0068 and 0.185-0.556. mu.M GDC-0973 (BLISS score ≧ 15). Thus, the combination of GDC-0068 and GDC-0973 also resulted in a synergistic effect on cell death induction in HCT-116 cells.

Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown and described. Accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention as defined by the claims that follow.

Claims (28)

1. A combination of a compound of formula I or a pharmaceutically acceptable salt thereof and a drug selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is:

the combination is useful for the prophylactic or therapeutic treatment of hyperproliferative diseases.

2. The combination of claim 1, wherein the hyperproliferative disease is cancer.

3. The combination of claim 2, wherein the cancer is associated with a PTEN mutation.

4. The combination of claim 2, wherein the cancer is associated with AKT mutation, overexpression or amplification.

5. The combination of claim 2, wherein the cancer is associated with a PI3K mutation.

6. The combination of claim 2, wherein the cancer is associated with Her2/ErbB2 amplification.

7. The combination of any one of claims 2-6, wherein the cancer is selected from mesothelioma, endometrial, pancreatic, breast, lung, ovarian, prostate, melanoma, gastric, colon, renal, head and neck, and glioma.

8. The combination of any one of claims 1-7, wherein a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973.

9. The combination of any one of claims 1-7, wherein a compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with PD-0325901.

10. The combination of any one of claims 1-9, wherein the compound of formula I or salt thereof and the one or more agents are administered simultaneously.

11. The combination of any one of claims 1-9, wherein the compound of formula I or salt thereof and the one or more agents are administered sequentially.

12. The combination of any one of claims 1-9, wherein administration of the one or more agents begins about 1 to about 10 days prior to administration of the combination.

13. The combination of any one of claims 1-9, wherein administration of the compound of formula I or salt thereof is initiated about 1 to about 10 days prior to administration of the combination.

14. The combination of any one of claims 1-9, wherein the compound of formula I or salt thereof and the one or more agents are administered beginning on the same day.

15. The combination of any one of claims 1-7, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973 and the cancer is pancreatic cancer.

16. The combination of any one of claims 1-7, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973 and the cancer is non-small cell lung cancer.

17. The combination of any one of claims 1-7, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973 and the cancer is breast cancer.

18. The combination of any one of claims 1-7, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973 and the cancer is colon cancer.

19. The combination of any one of claims 1-7, wherein the compound of formula I, or a pharmaceutically acceptable salt thereof, is administered in combination with GDC-0973 and the cancer is melanoma.

20. A compound of formula I or a pharmaceutically acceptable salt thereof, for therapeutic use in improving the quality of life of a patient treated for a hyperproliferative disease, together with an agent selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof.

21. A combination of a) a compound of formula I or a pharmaceutically acceptable salt thereof; and b) one or more drugs selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof for use in the treatment of a hyperproliferative disease.

22. A combination of a) a compound of formula I or a pharmaceutically acceptable salt thereof; and b) one or more drugs selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or condition modulated by AKT kinase.

23. Use of a combination of a compound of formula I or a pharmaceutically acceptable salt thereof and GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a hyperproliferative disease in a mammal.

24. Use of a combination of a compound of formula I or a pharmaceutically acceptable salt thereof and GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease or condition modulated by AKT kinase in a mammal.

25. A kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof, a container, and a package insert or label indicating administration of a compound of formula I and one or more agents selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof for use in the treatment of a hyperproliferative disease.

26. A product comprising a compound having formula I or a pharmaceutically acceptable salt thereof and one or more agents selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof; the product is used as a combined preparation for separate, simultaneous or sequential use in the treatment of hyperproliferative diseases.

27. A method for the treatment of a hyperproliferative disease in a mammal comprising administering to said mammal a combination of a compound of formula I:

28. a method for treating a disease or condition modulated by AKT kinase in a mammal, comprising administering to said mammal a) a compound of formula I or a pharmaceutically acceptable salt thereof; and b) one or more drugs selected from GDC-0973, PD-0325901, or a pharmaceutically acceptable salt thereof.