2-anilinopyrimidine derivatives as therapeutic agents for treatment of brain cancer

Abstract

The present invention relates to 2-anilinopyrimidine derivatives as therapeutic agents for the treatment of brain cancer. Specifically, the invention discloses a method for treating brain cancer, especially EGFR-mediated metastatic brain cancer, by using substituted 2-anilinopyrimidine derivatives, and pharmaceutically acceptable salts, solvates or compositions thereof.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780036188.7, filed on May 11, 2017, entitled “2-anilinopyrimidine derivatives as therapeutic agents for treating brain cancer”.

[0002] Cross-references to related patent applications

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 334,830, filed on May 11, 2016, the disclosure of which is incorporated herein by reference. Technical Field

[0004] The present invention relates to a method for treating EGFR-mediated metastatic brain cancer using 2-anilinopyrimidine derivatives and pharmaceutically acceptable salts and compositions thereof. Background Art

[0005] Epidermal growth factor receptor (EGFR, Her1, ErbB1) is the main member of the ErbB family of 4 structurally related cell surface receptors, the other members being Her2 (Neu, ErbB2), Her3 (ErbB3) and Her4 (ErbB4). EGFR exerts its major cellular function through its intrinsic catalytic tyrosine protein kinase activity. The receptor is activated by binding to growth factor ligands, such as epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), and the catalytically inactive EGFR monomers are transformed into catalytically active homopolymers and heterodimers. These catalytically active dimers then initiate intracellular tyrosine kinase activity, which leads to autophosphorylation of specific EGFR tyrosine residues and induces downstream activation of signaling proteins. Subsequently, signaling proteins initiate multiple signal transduction cascades (MAPK, Akt and JNK), ultimately regulating the fundamental biological processes of cell growth, proliferation, motility and survival.

[0006] EGFR is found at abnormally high levels on the surface of many types of cancer cells, and elevated EGFR levels have been associated with advanced disease, cancer spread, and poor clinical prognosis. Mutations in EGFR can lead to overexpression, permanent activation, or persistent hyperactivity of the receptor, leading to uncontrolled cell growth, i.e., cancer. As a result, EGFR mutations have been identified in several types of malignancies, including metastatic lung, head and neck, colorectal, and pancreatic cancers. In brain cancer, mutations occur primarily in exons 18-21, which encode the adenosine triphosphate (ATP) binding pocket of the kinase domain. The most clinically relevant drug-sensitive EGFR mutations are deletions in exon 19 and point mutations in exon 21, the former eliminating a common amino acid motif (LREA), while the latter results in a substitution of arginine by leucine at position 858 (L858R). Together, these two activating mutations account for nearly 85% of EGFR mutations observed in brain cancer. Both mutations have permanent tyrosine kinase activity, and are therefore oncogenic. In at least 50% of patients who initially respond to current therapies, disease progression is associated with the development of a secondary mutation, T790M in exon 20 of EGFR (also known as the gatekeeper mutation).

[0007] About 30-50% of patients with non-small cell lung cancer develop brain metastases (BM) (Baik, CS; J. Thorac. Oncol. 2015, 10, 1268), but there is currently no effective therapy for their treatment. Summary of the invention

[0008] In one aspect, the present invention provides a method of treating brain cancer in an individual, comprising administering to the individual a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof:

[0009]

[0010] in:

[0011] R 1 is selected from hydrogen, halogen, methyl, trifluoromethyl and cyano;

[0012] R 2 , R 3 and R 4 are the same or different and independently selected from hydrogen, halogen and trifluoromethyl;

[0013] R 5 is selected from lower alkyl, optionally substituted 3-6 membered heterocyclic group, R 7 R 8 N-(lower alkyl), and R 7 R 8 N-(cycloalkylalkyl), where R7 and R 8 are the same or different and are independently selected from hydrogen and lower alkyl;

[0014] R 6 is selected from lower alkoxy and lower alkyl;

[0015] Q is for CR 10 or N;

[0016] R 9 It is C 1 -C 4 Alkyl or C 1 -C 4 haloalkyl; and

[0017] R 10 is H or CH 3 .

[0018] In a preferred embodiment, the compound of formula (I) is Compound 1.

[0019] In another preferred embodiment, the brain cancer is metastatic brain cancer, and more preferably is metastatic brain cancer developed from non-small cell lung cancer mediated by EGFR.

[0020] The efficacy of compound 1 in mice was determined by observing tumor regression of implanted tumors from the luciferase-labeled NCI-H1975 human cell line. Efficacy was based on bioluminescent imaging (BLI) data combined with traditional survival endpoints. An increase in survival of more than 100% was observed in animals treated with compound 1 compared to animals treated with vehicle.

[0021]

[0022] Other aspects or advantages of the present invention will be reflected in the following figures, detailed description of the embodiments and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Comparison of brain tumor BLI signal groups between treatment with 1·Ms and vehicle control is shown.

[0024] Figure 2 The percentage survival between groups is shown, comparing 1·Ms treatment and vehicle control. Specific implementation plan

[0025] In one aspect, the present invention provides a method of treating brain cancer in an individual, comprising administering to the individual a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, or composition thereof:

[0026]

[0027] in:

[0028] R 1 is selected from hydrogen, halogen, methyl, trifluoromethyl and cyano;

[0029] R 2 , R 3 and R 4 are the same or different and independently selected from hydrogen, halogen and trifluoromethyl;

[0030] R 5 is selected from lower alkyl, optionally substituted 3-6 membered heterocyclic group, R 7 R 8 N-(lower alkyl) and R 7 R 8 N-(cycloalkylalkyl), where R 7 and R 8 are the same or different and are independently selected from hydrogen and lower alkyl;

[0031] R 6 is selected from lower alkoxy and lower alkyl; and

[0032] Q is for CR 10 or N;

[0033] R 9 It is C 1 -C 4 Alkyl or C 1 -C 4 haloalkyl; and

[0034] R 10 is H or CH 3 .

[0035] In one embodiment of this aspect, in the compound of formula (I), Q is CR 10 .

[0036] In another embodiment of this aspect, in the compound of formula (I), R 5 Selected from C 1 -C 6 alkyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted piperidinyl, R 7 R 8 N-(CH 2 ) n -(n=1-5), R 7 R 8 N-(C 3 -C 6 Cycloalkyl)-(CH 2 ) m-(m=1-3), where R 7 and R 8 are the same or different and are independently selected from hydrogen and lower alkyl.

[0037] In another embodiment of this aspect, in the compound of formula (I), R 5 Selected from methyl, R 7 R 8 N-(CH 2 ) n -(n=2 or 3), 1-(dimethylamino)-cyclopropylmethyl, 3-(dimethylamino)cyclobutyl, 1-methylazetidin-3-yl, (R)-1-methylpyrrolidin-3-yl, (S)-1-methylpyrrolidin-3-yl, and 1-methylpiperidin-4-yl.

[0038] In another embodiment of this aspect, in the compound of formula (I), R 5 It is 2-dimethylamino-ethyl [(CH 3 ) 2 NCH 2 CH 2 -].

[0039] In another embodiment of this aspect, in the compound of formula (I), R 1 is hydrogen or halogen or methyl.

[0040] In another embodiment of this aspect, in the compound of formula (I), R 1 It's hydrogen.

[0041] In another embodiment of this aspect, in the compound of formula (I), R 2 is hydrogen or a halogen.

[0042] In another embodiment of this aspect, in the compound of formula (I), R 4 It's hydrogen.

[0043] In another embodiment of this aspect, in the compound of formula (I):

[0044] R 2 is hydrogen, F or Cl;

[0045] R 3 is hydrogen, F, Cl or -CF 3 ;as well as

[0046] R 4 It's hydrogen.

[0047] In another embodiment of this aspect, in the compound of formula (I), R 1 , R 2 , R 3and R 4 It's all hydrogen.

[0048] In another embodiment of this aspect, the compound of formula (I) is further characterized by the structure of formula (II):

[0049]

[0050] in:

[0051] Q is for CR 10 or N;

[0052] R 9 Yes CH 3 or CH 2 CH 2 F; and

[0053] R 10 is H or CH 3 .

[0054] In another embodiment of this aspect, in the compound of formula (II), Q is CR 10 .

[0055] In another embodiment of this aspect, in the compound of formula (II), R 9 Yes CH 3 .

[0056] In another embodiment of this aspect, in the compound of formula (II), Q is CH.

[0057] In a preferred embodiment of the present invention, the compound of formula (I) is further characterized by the structure of the following formula:

[0058]

[0059] In another preferred embodiment of this aspect, the compound of formula (I) is a pharmaceutically acceptable salt of compound 1. In another preferred embodiment, the compound of formula (I) is a mesylate salt of compound 1, i.e., 1·Ms.

[0060]

[0061] In another embodiment of this aspect, the compound of formula (I) is further characterized by the structure of the following formula:

[0062]

[0063] In another embodiment of this aspect, in the compound of formula (II), R 9 Yes CH 2 CH 2 F.

[0064] In another embodiment of this aspect, the compound of formula (I) is further characterized by the structure of the following formula:

[0065]

[0066] In another embodiment of this aspect, the compound of formula (I) is selected from the following group:

[0067]

[0068]

[0069]

[0070] In another embodiment of this aspect, the compound of formula (I) is selected from the following group:

[0071]

[0072] In another embodiment of this aspect, the method further comprises administering to the individual a second therapeutic agent.

[0073] In another embodiment of this aspect, the second therapeutic agent is a different EGFR modulator.

[0074] In another embodiment of this aspect, the second therapeutic agent is a chemotherapeutic drug.

[0075] In another embodiment of this aspect, the brain cancer is metastatic brain cancer.

[0076] In a preferred embodiment of this aspect, the brain cancer is a metastatic brain cancer mediated by EGFR cancer development.

[0077] In another preferred embodiment of the present invention, the brain cancer is metastatic brain cancer developed from non-small cell lung cancer mediated by EGFR.

[0078] In another embodiment of this aspect, the method according to any one of the above embodiments comprises administering to the subject a pharmaceutical composition comprising a compound of formula (I) or (II), or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier.

[0079] In another embodiment of this aspect, the compound of formula (I) or (II) is Compound 1.

[0080] In another embodiment of this aspect, the compound of formula (I) or (II) is the mesylate salt of Compound 1 (1·Ms).

[0081] In another aspect, the present invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, or composition thereof, in the preparation of a medicament for treating brain cancer:

[0082]

[0083] in:

[0084] R 1 is selected from hydrogen, halogen, methyl, trifluoromethyl and cyano;

[0085] R 2 , R 3 and R 4 are the same or different and independently selected from hydrogen, halogen and trifluoromethyl;

[0086] R 5 is selected from lower alkyl, optionally substituted 3-6 membered heterocyclic group, R 7 R 8 N-(lower alkyl), and R 7 R 8 N-(cycloalkylalkyl), where R 7 and R 8 are the same or different and are independently selected from hydrogen and lower alkyl;

[0087] R 6 is selected from lower alkoxy and lower alkyl; and

[0088] Q is for CR 10 or N;

[0089] R 9 It is C 1 -C 4 Alkyl or C 1 -C 4 haloalkyl; and

[0090] R 10 is H or CH 3 .

[0091] In one embodiment of this aspect, the compound of formula (I) is further characterized by the structure of formula II below:

[0092]

[0093] in:

[0094] Q is for CR 10 or N;

[0095] R 9 Yes CH 3 or CH 2 CH 2F; and

[0096] R 10 is H or CH 3 .

[0097] In another embodiment of this aspect, the compound of formula (I) is selected from the following group:

[0098]

[0099] In another embodiment of this aspect, the brain cancer is metastatic brain cancer.

[0100] In another embodiment of this aspect, the brain cancer is metastatic brain cancer developed from EGFR-mediated non-small cell lung cancer.

[0101] Other aspects or embodiments of the invention include substantially as shown and described herein and any possible combination of any two or more embodiments described herein.

[0102] Terms in this application, unless otherwise specifically defined, have their common meanings as understood by those skilled in the art.

[0103] As used herein, the term "halo" or "halogen" refers to F, Cl or Br.

[0104] The term "lower alkyl" refers to a branched or straight chain alkyl group having 1 to 7, preferably 1 to 4, and more preferably 1 to 2 carbon atoms.

[0105] The term "lower alkoxy" refers to an alkoxy group (-OR) having 1 to 7, preferably 1 to 4, and more preferably 1 to 2 carbon atoms.

[0106] The term "cyano" refers to -CN.

[0107] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response or other problems or complications commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0108] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art, such as ion exchange, etc. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

[0109] The term "solvate" as used herein refers to a stoichiometric or non-stoichiometric physical combination of a compound of the present invention and a solvent molecule. For example, one molecule of the compound is combined with one or more, preferably 1-3, solvent molecules. It is also possible that multiple (e.g., 2) compound molecules share one solvent molecule. This physical combination may include hydrogen bonds. In some cases, the solvate can be isolated as a crystalline solid. The solvent molecules in the solvate may exist in a regular arrangement and / or a non-ordered arrangement. Typical solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are generally known in the art.

[0110] The term "prodrug", as used herein, refers to a derivative of a compound that can be converted in vivo to the parent compound, for example by hydrolysis in the blood. Common examples include, but are not limited to, ester and amide forms of active carboxylic acid compounds; or vice versa, esters of active alcohol compounds or amide forms of active amine compounds. These amide or ester prodrug compounds can be prepared according to conventional methods known in the art. For example, the prodrug of the compound of formula II of the present invention can be in the form of the following formula III:

[0111]

[0112] Where R x and R y are independently H and -C(O)-R, wherein R is C 1 -C 4 Alkyl, preferably methyl or ethyl, and more preferably methyl. Other prodrugs of the present invention can be prepared similarly.

[0113] When used in treatment, a therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, may be used directly as a raw material, and the active ingredient may be presented as a pharmaceutical composition. Therefore, the present disclosure further provides a pharmaceutical composition, which includes any compound of the present invention or a pharmaceutically acceptable salt or solvate thereof, and one or more, preferably 1-3 pharmaceutically acceptable carriers, diluents or other excipients. The carrier, diluent or other excipient must be acceptable in the sense of being compatible with the other ingredients of the preparation and not harmful to the individual receiving the treatment.

[0114] Pharmaceutical preparations can be presented as unit dose preparations, each unit dose containing a predetermined amount of active ingredient. Typically, the pharmaceutical composition of the present disclosure will be administered about 1 to about 5 times a day, or by continuous infusion. This administration can be used as a chronic or acute therapy. The amount of active ingredient combined with a carrier material to produce a single dose preparation can vary according to the disease to be treated, the severity of the disease, the time of administration, the route of administration, the excretion rate of the compound used, the duration of treatment, and the age, sex, weight, and condition of the patient. Preferred unit dose preparations are those containing a daily dose or subdose, as described above, or an appropriate portion, active ingredient. Typically, treatment begins with a smaller dose that is much less than the optimal dose of the compound. Thereafter, the dosage is increased in small amounts until the optimal effect in this case is achieved. In general, the most desirable administration of the compound is a concentration level that generally obtains effective results without causing substantial harm or harmful side effects.

[0115] When the compositions of the present disclosure comprise a combination of a compound of the present disclosure and one or more, preferably one or two, additional therapeutic or prophylactic agents, both the compound and the additional active agent are typically present at dosage levels of about 10 to 150%, more preferably about 10 to 80%, of the dosage normally administered as a monotherapy regimen.

[0116] The pharmaceutical preparation may be suitable for administration by any suitable route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal, or parenteral (including subcutaneous, intradermal, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous and intradermal injection or infusion) routes. Such preparations may be prepared by any method known in the pharmaceutical art, for example, by combining the active ingredient with one or more carriers or excipients. Oral administration or administration by injection is preferred.

[0117] Pharmaceutical formulations suitable for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or thick foams; or oil-in-water liquid emulsions or water-in-oil emulsions.

[0118] For example, for oral administration in the form of tablets or capsules, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Powders are prepared by grinding the compound to a suitable fine size and mixing with a similarly ground pharmaceutical carrier, such as an edible carbohydrate, for example starch or mannitol. Flavoring agents, preservatives, dispersants and coloring agents may also be present.

[0119] Capsules are prepared by preparing a powder mixture as described above and filling it into a shaped gelatin shell. Glidants and lubricants such as colloidal silicon dioxide, talc, magnesium stearate, calcium stearate or solid polyethylene glycol may be added to the powder mixture before the filling operation. Disintegrants or solubilizers such as agar, calcium carbonate or sodium carbonate may also be added to improve the availability of the drug when the capsule is ingested.

[0120] In addition, when necessary or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like. Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like. Disintegrants include but are not limited to starch, methylcellulose, agar, bentonite, xanthan gum and the like. For example, tablets are prepared by preparing a powder mixture, granulating or pre-pressing, adding lubricants and disintegrants and pressing into tablets. The powder mixture is prepared as follows: mixing the appropriately crushed compound with a diluent or base ingredient as described above, and optionally with a binder such as carboxymethylcellulose, alginate, gelatin or polyvinyl pyrrolidone, a solution retardant such as paraffin, a reabsorption accelerator such as a quaternary salt and / or with an absorbent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be wetted with a binder such as syrup, starch paste, acacia gum or a solution of cellulose or polymeric material and forced through a screen for granulation. As an alternative to the granulation method, the powder mixture can be passed through a tablet press, resulting in the incompletely formed pre-compressed tablets being broken into granules. The granules can be lubricated by adding stearic acid, stearate, talc or mineral oil to prevent sticking to the tablet forming die. The lubricated mixture is pressed into tablets. The compounds of the present disclosure can also be mixed with a free-flowing inert carrier and directly pressed into tablets without the need for granulation or pre-compression steps. A transparent or opaque protective coating consisting of a shellac sealing coating, a sugar or polymeric material coating, and a wax polishing coating can be provided. Dyes can be added to these coatings to distinguish different unit doses.

[0121] Oral liquids such as solutions, syrups and elixirs can be prepared in dosage unit form so that a given amount contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in an appropriately flavored aqueous solution, while elixirs are prepared by using a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservatives, flavoring additives such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, etc. can also be added.

[0122] Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The preparation can also be prepared to prolong or sustain the release, for example, by coating or embedding particulate material in polymers, wax or the like.

[0123] It should be understood, however, that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0124] The term "patient" or "subject" includes humans and other mammals.

[0125] The term "mammal" or "mammals" includes, but is not limited to, humans, dogs, cats, horses, pigs, cows, monkeys, rabbits and mice. The preferred mammal is a human.

[0126] The term "therapeutically effective amount" refers to an amount of a compound or composition that, when administered to an individual to treat a disease, is sufficient to achieve the treatment for the disease. The "therapeutically effective amount" may vary depending on, inter alia, the compound, the disease and its severity, and the age, weight, or other factors of the patient being treated. When applied to an individual active ingredient administered alone, the term refers to that individual ingredient. When applied to a combination, the term refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.

[0127] The term "treat" (verb) or "treat" (noun) means: (i) inhibiting a disease, disorder or condition, i.e., arresting its development; (ii) alleviating a disease, disorder or condition, i.e., causing regression of the disease, disorder and / or condition; or (iii) preventing a disease, disorder or condition from occurring in an individual who may be susceptible to the disease, disorder and / or condition but has not yet been diagnosed as having it. Thus, in one embodiment, "treat" (verb) or "treat" (noun) refers to ameliorating a disease or condition, which may include ameliorating one or more physical parameters, although the individual being treated may not be discernible. In another embodiment, "treat" (verb) or "treat" (noun) includes modulating a disease or condition, either physically (e.g., stabilization of discernible symptoms) or physiologically (e.g., stabilization of a physical parameter) or both. In another embodiment, "treat" (verb) or "treat" (noun) includes delaying the onset of a disease or condition.

[0128] When the term "about" is applied to a parameter, such as content, temperature, time or the like, it indicates that the parameter can generally vary by ±10%, preferably within ±5%, and more preferably within ±2%. As will be appreciated by those skilled in the art, when a parameter is not critical, the numbers provided in the examples are generally given only for illustrative purposes and are not limiting.

[0129] As used herein, the terms "a", "an" or "the" refer to both the singular and plural forms. Generally, when a singular or plural form of a noun is used, it refers to both the singular and plural forms of the noun.

[0130] The following non-limiting examples further illustrate certain aspects of the present invention.

[0131] Example

[0132] Materials and methods

[0133] N-(2-(2-(dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide methanesulfonate (1·Ms; C27H30N6O3·1.06CH3SO3H, MW=486.58, FW=588.45, purity=100%) was formulated in a vehicle of 1% Tween 80 in water. All vehicle was added to the pre-weighed compound to give a 5 mg / mL stock solution, suitable for treatment at a 50 mg / kg dose level. The mixture was vortexed for approximately 1 minute to produce a pale yellow solution with a pH of 7. The dosing solution was prepared fresh daily.

[0134] animal

[0135] Female Envigo Nude mice (Hsd: Athymic Nude-Foxn1nu) were used. They were 6-7 weeks old on day 1 of the experiment. The animals were fed with irradiated Harlan 2918.15 Rodent Diet and water ad libitum. The animals were placed in static cages with a fixed Bed-O'Cobs in Clean Rooms TM , which provides HEPA filtered air to the bubble environment, with 100 complete air changes per hour. All treatments, body weight determinations, and tumor measurements are performed in the bubble environment. The environment is controlled at a temperature of 70±2°F and a humidity of 30-70%.

[0136] Cell preparation

[0137] NCI-H1975-Luc cells were obtained from Clovis. They were grown in RPMI 1640 culture medium modified with the following substances: 1 mM sodium pyruvate + 2 mM L-glutamine + 10 mM HEPES + 2.5 g / L glucose + 5 μg / mL blasticidin, and supplemented with 10% non-heat-inactivated fetal bovine serum (FBS) and 1% 100X penicillin / streptomycin / L-glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO2 atmosphere at 37°C. When expansion was complete, the cells (passage 4) were trypsinized with 0.25% trypsin-EDTA solution. After cell separation, dilute with complete growth medium, thereby inactivating trypsin, and then separate any cell clumps by pipetting. The cells were centrifuged at 200rcf for 8 minutes at 4°C, the supernatant was removed, and the precipitate was then resuspended in cold Dulbecco's phosphate buffered saline (DPBS) by pipetting. The uniform cell suspension of equal portions was diluted in trypan blue solution, and Luna automatic cell counter was used to count, to determine cell viability before transplantation. The cell suspension was centrifuged at 200rcf for 8 minutes at 4°C. The supernatant was removed, and the cell pellet was resuspended in cold serum-free medium, to produce a final concentration of 1.00E+08 trypan blue-free cells / mL. The cell suspension was maintained on wet ice during transplantation. After transplantation, the residual cells of equal portions were diluted and counted with trypan blue solution, to determine cell viability after transplantation.

[0138] Intracranial transplantation

[0139] According to the treatment plan (Appendix 1), the test mice were transplanted intracranially with 1.00E+06 cells / 10 μL on day 0. For aseptic surgical transplantation, mice were injected with 0.2 mg / kg buprenorphine and anesthetized with 2% isoflurane in air. The mice were then fixed on a stereotaxic frame (ASI instruments, Inc.) with non-ruptured ear bars. Eye ointment was applied to the eyes of the mice to prevent dryness during surgery. During the transplantation process, a recirculating 37°C water heating pad was used to maintain the body temperature of the animals. Once in the stereotaxic frame, the skull was wiped with a chlorhexidine solution and a swab saturated with 70% ethanol in turn to disinfect the skin surface and prepare the incision. A 1 cm long incision was made along the center on the crown sagittal point of the skull using a #15BD surgical blade. The incision was retracted with a small serrated vascular clamp. The thin connective tissue layer covering the surface of the skull was removed with a dry cotton swab under light pressure. The blood vessels were cauterized to prevent blood loss. A 0.9mm drill was placed centered on the coronal sagittal point, moved 2mm to the right, 1mm in front of the coronal suture, and then lowered with a stereotaxic electrode operating arm to mark the surface of the skull. The drill was removed from the stereotaxic frame, and the drilling from the skull to the dura mater surface was completed manually. The cell suspension (stored on wet ice) was fully mixed and then drawn into a gas-sealed 50μL syringe. A standard 27g needle was filled with the cell suspension to eliminate bubbles, and then the luer tip of the syringe was inserted into the needle interface. The syringe was fixed on a custom syringe holder (ASI Instruments, Inc.) and connected to the stereotaxic frame operating arm. The syringe needle was centered on the drill hole and lowered to the level of the lower side of the skull at the tilted head and the dura mater surface. The needle then dropped 3mm in the brain and then retracted 1mm to form a "reservoir" for depositing the cell suspension. Then 10μL of the cell suspension (1x10 6 Cells / mouse) were injected into the brain tissue, with any minor leaks (usually IC grafts) being absorbed with dry cotton swabs. After injection, the needle was withdrawn and the burr hole was immediately sealed with bone wax to minimize the loss of transplanted cells. The skull surface was cleaned with dry cotton swabs and cotton swabs saturated with 70% ethanol in turn to remove external cells and prevent tumor growth outside the skull. The mouse was removed from the stereotaxic frame and the incision was closed with stainless steel wound clips. Once the mouse regained consciousness and lay on its back, it was returned to the cage for breeding.

[0140] treat

[0141] All mice were divided into treatment groups based on the estimation of tumor burden by bioluminescent imaging. The distribution of these mice ensured that the mean tumor burden in all groups was within 10% of the total mean tumor burden of the study mouse population. Treatment started on day 5.

[0142] Group 1: Vehicle control (1% Tween 80), 0.2 mL / 20 g, PO, QDx11 (once a day from day 5 to day 15)

[0143] Group 2: 1·Ms, 50 mg / kg, PO, QDx14 (once a day from day 5 to day 18)

[0144] Bioluminescence imaging

[0145] In vivo bioluminescent imaging was performed using an IVIS 50 optical imager (Xenogen, Alameda, CA). Animals were imaged 3 at a time under ~1-2% isoflurane gas anesthesia. Each mouse was injected with 150 mg / kg D-luciferin IP and imaged in the prone position 10 minutes after the injection. A large binning CCD chip was used, and the exposure time (2 seconds-2 minutes) was adjusted to obtain at least hundreds of counts of metastatic tumors observable in each mouse in the image, and to avoid saturation of the CCD chip. BLI images were collected on days 5, 8, 11, 13, 15, and 18. These images were analyzed using the software Living Image version 4.3.1 (Xenogen, Alameda, CA). For each individual animal, a fixed-size ROI was placed to include the in situ tumor on the prone image and marked based on the animal identity. The total flux (photons / second) was calculated and extrapolated for all ROIs to facilitate inter-group analysis.

[0146] Measurements and Endpoints

[0147] The %T / C of the original tumor burden (estimated by BLI) was used as the original endpoint in this study. %T / C is defined as follows: the median BLI signal of the treatment group is divided by the median BLI signal of the control group × 100. The %T / C on the 13th day was used for analysis because it was the last day for imaging and the median of the animals that remained in the study more than the control group. Extended survival was used as a secondary endpoint in this study. Complete response (CR) was defined as a decline in tumor weight (based on bioluminescent imaging) to an unreliable signal (less than 2.0E+05 photons / second). The background level of BLI is usually 1E+03-1E+04 photons / second. Partial response (PR) is defined as a reduction of ≥50% in tumor BLI signal from the first treatment. PR does not include CR.

[0148] Effectiveness Results

[0149] Group 2: 1·Ms, 50 mg / kg, PO, QDx14 (once a day from day 5 to day 18)

[0150] Based on BLI %T / C (1%, Figure 1 ), treatment with 1·Ms produced significant (P<0.05) anticancer activity. The median survival was 29.0 days (107% ILS or 15 days of increased survival) (p<0.001). By day 18, 100% of the animals had partial tumor regression, but none had complete regression ( Figure 2 ).

[0151] In addition to the activating mutation, compound 1 also effectively inhibits the kinase domain of the T790M double mutant and thus overcomes the resistance observed in the therapy with currently used reversible inhibitors. Because the role of EGFR in non-small cell lung cancer (NSCLC) is known (Ohashi, K. et al.; J. Clin. Oncol. 2013, 31, 1070), 1 represents a potential therapeutic drug that can be used to treat non-small cell lung cancer.

[0152] When orally administered in the rats, compound 1 reached therapeutic brain concentrations (Table 1). In addition, 1 was effective against brain tumors implanted intracranially in mice. Therefore, 1 represents a potential therapeutic drug for the treatment of EGFR-mediated metastatic brain cancer.

[0153] Treatment with 1·Ms was well tolerated and produced significant (P<0.05) anticancer activity based on BLI %T / C and survival at day 13. Although all mice eventually succumbed to disease, treatment with 1·Ms more than doubled survival.

[0154] Table 1. 1·Ms pharmacokinetic (PK) parameters in male SD rats dosed PO@5 mg / kg with 0.5% methylcellulose vehicle

[0155] parameter plasma brain <![CDATA[C max (ng / mL or ng / g)]]> 96.3 606 <![CDATA[T max (h)]]> 2.00 2.00 <![CDATA[T 1 / 2 (h)]]> 1.97 2.01 <![CDATA[T last (h)]]> 18.0 18.0 <![CDATA[AUC 0-last (ng·h / mL or ng·h / g)]]> 535 5047 <![CDATA[AUC 0-inf (ng·h / mL or ng·h / g)]]> 538 5085 <![CDATA[MRT 0-last (h)]]> 5.01 5.95 <![CDATA[MRT 0-inf (h)]]> 5.10 6.07 <![CDATA[AUC Extra (%)]]> 0.593 0.746 <![CDATA[AUMC Extra (%)]]> 2.42 2.57 <![CDATA[ d AUC Ratio]]> -- 9.44

[0156] The above examples or preferred embodiments are provided for illustrative purposes and are not intended to limit the present invention. Various changes and combinations of the above features may be utilized without departing from the present invention as defined in the claims.

Claims

1. Use of a compound of formula 2, or a pharmaceutically acceptable salt, solvate, prodrug or composition thereof, in the preparation of a drug for treating metastatic brain cancer, wherein: The compound of formula 2 is characterized by the following structure:

2. Use of a compound of formula 3, or a pharmaceutically acceptable salt, solvate, prodrug or composition thereof, in the preparation of a drug for treating metastatic brain cancer, wherein: The compound of formula 3 is characterized by the following structure, wherein the compound of formula II is further characterized by the following structure:

3. The use according to claim 1 or 2, wherein a second therapeutic agent is used in combination.

4. The use of claim 3, wherein the second therapeutic agent is a different EGFR modulator.

5. The use of claim 3, wherein the second therapeutic agent is a chemotherapeutic drug.

6. The use according to any one of claims 1 to 5, wherein the metastatic brain cancer is a metastatic brain cancer developed from non-small cell lung cancer mediated by EGFR.

7. The use according to any one of claims 1 to 5, comprising administering to an individual a pharmaceutical composition comprising the compound of Formula 1 or Formula 2, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier.

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