OA19269A - 2-anilinopyrimidine derivatives as therapeutic agents for treatment of brain cancers. - Google Patents

Abstract

Methods of using substituted 2-anilinopyrimidine derivatives, and pharmaceutically acceptable salts, solvates, or compositions, for the treatment of brain cancers, in particular EGFR-mediated metastatic brain cancer, are disclosed.

Description

2-ANILINOPYRIMIDINE DERIVATIVES AS THERAPEUTIC AGENTS FOR TREATMENT OF BRAIN CANCERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application daims priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62/334, 830, filed May 11, 2016, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The présent invention relates to a method of treating EGFR-mediated metastatic brain cancer with 2-anilinopyrimidine dérivatives, and pharmaceutically acceptable salts and compositions thereof.

BACKGROUND OF THE INVENTION

The epidermal growth factor receptor (EGFR, Herl, ErbBl) is a principal member of the ErbB family of four structurally-related cell surface receptors with the other members being Her2 (Neu, ErbB2), Her3 (ErbB3) and Her4 (ErbB4). EGFR exerts its primary cellular functions though its intrinsic catalytic tyrosine protein kinase activity. The receptor is activated by binding with growth factor ligands, such as epidermal growth factor (EGF) and / transforming growth factor-alpha (TGF-α), which transform the catalytically inactive EGFR monomer into catalytically active homo- and hetero- dimers. These catalytically active dimers then initiate intracellular tyrosine kinase activity, which leads to the autophosphorylation of spécifie EGFR tyrosine residues and elicits the downstream activation of signaling proteins. Subsequently, the signaling proteins initiate multiple signal transduction cascades (MAPK, Akt and JNK), which ultimately médiate the essential biological processes of cell growth, prolifération, mptility and survival.

EGFR is found at abnormally high levels on the surface of many types of cancer cells and increased levels of EGFR hâve been associated with adyanced disease, cancer spread and poor clinical prognosis. Mutations in EGFR can lead to receptor overexpression, perpétuai activation or sustained hyperactivity and resuit in uncontrolled cell growth, i.e. cancer. Consequently, EGFR mutations hâve been identified in several types of malignant tumors, including metastatic lung, head and neck, colorectal and pancreatic cancers. In lung cancer, mutations mainly occur in exons 18 to 21, which encode the adenosine triphosphate (ATP)binding pocket of the kinase domain. The most clinically relevant drug-sensitive EGFR

A mutations are délétions in exon 19 that eliminate a common amino acid motif (LREA) and point mutations in exon 21, which lead to a substitution of arginine for leucine at position 858 (L858R). Together, these two activating mutations account for nearly 85% of the EGFR mutations observed in lung cancer. Both mutations hâve perpétuai tyrosine kinase activity and as a resuit they are oncogenic. In at least 50% of patients who are initially responsive to current therapy, disease progression is associated with the development of a secondary mutation, T790M in exon 20 of EGFR (referred to as the gatekeeper mutation).

Approximately 30-50% of non-small cell lung cancer patients develop brain métastasés (BM) (Baik, C.S.; J. Thorac. Oncol. 2015, 10, 1268), but currently no effective therapy is available for their treatment.

SUMMARY OF THE INVENTION

The présent invention, in one aspect, provides a method of treating brain cancers in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):

(I) or a pharmaceutically acceptable sait, solvaté or prodrug thereof, wherein:

R* is selected from hydrogen, halogen, methyl, trifluoromethyl, and cyano;

R², R³, and R⁴ are the same or different and are independently selected from hydrogen, halogen, and trifluoromethyl;

R is selected from lower alkyl, optionally substituted 3- to 6-membered heterocyclyl, R⁷R⁸N-(lower alkyl), and R⁷R⁸N-(cycloalkylalkyl), wherein R⁷ and R⁸ are the same or different and are independently selected from hydrogen and lower alkyl;

R⁶ is selected from lower alkoxy and lower alkyl; and

Q is C-R¹⁰ or N

R⁹ is C1-C4 alkyl or C1-C4 haloalkyl; and

R¹⁰ is H or CH₃.

In a preferred embodiment, the compound of formula (I) is compound 1.

In another preferred embodiment, the brain cancer is a metastatic brain cancer, and more preferably a metastatic brain cancer developed from an EGFR-mediated non-small cell lung cancer.

Efficacy of 1 in mice was determined by observing tumor régression of intracranially implanted tumors from a luciferase-enabled NCI-H1975 human cell line. Efficacy was based on bioluminescence imaging (BLI) data coupled with traditional survival endpoints. A lifespan increase of greater than 100% was observed for animais treated with 1 vs. those treated with vehicle.

Other aspects or benefits of the présent invention will be reflected in the following drawings, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrâtes Brain Tumor BLI Signal Group comparison between treatment with 1-Ms and a vehicle control.

FIG. 2 illustrâtes percent survival rates by group, comparing 1-Ms treatment with a vehicle control.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the présent invention provides a method of treating brain cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):

or a pharmaceutically acceptable sait, solvaté, prodrug, or composition thereof, wherein:

R¹ is selected from hydrogen, halogen, methyl, trifluoromethyl, and cyano;

R², R³, and R⁴ are the same or different and are independently selected from hydrogen, halogen, and trifluoromethyl;

R is selected from lower alkyl, optionally substituted 3- to 6-membered heterocyclyl, R⁷R⁸N-(lower alkyl), and R⁷R⁸N-(cycloalkylalkyl), wherein R⁷ and R⁸ are the same or different and are independently selected from hydrogen and lower alkyl;

R⁶ is selected from lower alkoxy and lower alkyl; and

Q is C-R¹⁰ or N

R⁹ is C1-C4 alkyl or C1-C4 haloalkyl; and

R¹⁰ is H or CH₃.

In one embodiment of this aspect, in the compound of formula (I), Q is C-R¹⁰.

In another embodiment of this aspect, in the compound of formula (I), R⁵ is selected from Ci-C6 alkyl, substituted or unsubstituted azetidinyl, substituted or unsubstituted pyrrolidinyl, substituted or unsubstituted piperidinyl, R⁷R⁸N-(CH2)_n- (η = 1 to 5), R⁷R⁸N-(C₃Cé cycloalkyl)-(CH₂)_m- (m = 1 to 3), wherein R⁷ and R⁸ are the same or different and are independently selected from hydrogen and lower alkyl.

In another embodiment of this aspect, in the compound of formula (I), R⁵ is selected from methyl, R⁷R⁸N-(CH₂)_n- (n = 2 or 3), l-(dimethylamino)-cyclopropylmethyl, 3(dimethylamino)cyclobutyl, l-methylazetidin-3-yl, (7?)-l-methylpyrrolidin-3-yl, (5)-1methylpyrrolidin-3-yl, and l-methylpiperidin-4-yl.

In another embodiment of this aspect, in the compound of formula (I), R⁵ is 2dimethylamino-ethyl [(CH₃)₂NCH₂CH₂-].

In another embodiment of this aspect, in the compound of formula (I), R¹ is hydrogen or halogen, or methyl.

In another embodiment of this aspect, in the compound of formula (I), R¹ is hydrogen.

In another embodiment of this aspect, in the compound of formula (I), R² is hydrogen or halogen.

In another embodiment of this aspect, in the compound of formula (I), R⁴ is hydrogen.

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

R is hydrogen, F, or Cl;

R³ is hydrogen, F, Cl, or -CF₃; and

R⁴ is hydrogen.

In another embodiment of this aspect, in the compound of formula (I), R¹, R², R³, and R⁴ are ail hydrogen.

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

wherein:

Qis C-R¹⁰ or N

R⁹ is CH₃ or CH₂CH₂F; and

R¹⁰ is H or CH₃.

In another embodiment of this aspect, in the compound of formula (II), Q is C-R¹⁰.

In another embodiment of this aspect, in the compound of formula (II), R⁹ is CH₃.

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

In a preferred embodiment of this aspect, the compound of formula (I) is further characterized by a structure of formula:

In another preferred embodiment of this aspect, the compound of formula (I) is a pharmaceutically acceptable sait of the compound 1.

In another preferred embodiment of this aspect, the compound of formula (I) is a methanesulfonic acid sait of the compound 1, i.e., 1-Ms.

Ι-Ms

In another embodiment of this aspect, the compound of formula (I) is further characterized by a structure of formula:

In another embodiment of this aspect, in the compound of formula (II), R⁹ is CH₂CH₂F.

In another embodiment of this aspect, the compound of formula (I) is further characterized by a structure of formula:

F

In another embodiment of this aspect, the compound of formula (I) is selected from the group consisting of:

F

In another embodiment of this aspect, the method further comprises administering to the subject a second therapeutic agent.

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

In another embodiment of this aspect, the second therapeutic agent is a chemotherapeutic agent.

In another embodiment of this aspect, said brain cancer is a metastatic brain cancer.

In a preferred embodiment of this aspect, said brain cancer is a metastatic brain cancer developed from an EGFR-mediated cancer.

In another preferred embodiment of this aspect, said brain cancer is a metastatic brain cancer developed from an EGFR-mediated non-small cell lung cancer.

In another embodiment of this aspect, the method according to any embodiment described above comprises administering to the subject a pharmaceutical composition comprising said compound of formula (I) or (II), or a pharmaceutically acceptable sait, solvaté, or prodrug thereof, and a pharmaceutically acceptable carrier.

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

In another embodiment of this aspect, the compound of formula (I) or (II) is the methanesulfonic acid sait of compound 1 (1-Ms).

In another aspect, the présent invention provides use of a compound of formula (I), or a pharmaceutically acceptable sait, solvaté, prodrug, or composition thereof, in the manufacture of a médicament for the treatment of brain cancer:

(I) or a pharmaceutically acceptable sait, solvaté, prodrug, or composition thereof, wherein:

R¹ is selected from hydrogen, halogen, methyl, trifluoromethyl, and cyano;

R², R³, and R⁴ are the saine or different and are independently selected from hydrogen, halogen, and trifluoromethyl;

R⁵ is selected from lower alkyl, optionally substituted 3- to 6-membered heterocyclyl, R⁷R⁸N-(lower alkyl), and R⁷R⁸N-(cycloalkylalkyl), wherein R⁷ and R⁸ are the same or different and are independently selected from hydrogen and lower alkyl;

R⁶ is selected from lower alkoxy and lower alkyl; and

Q is C-R¹⁰ or N

R⁹ is C1-C4 alkyl or C1-C4 haloalkyl; and

R¹⁰ is H or CH₃.

In one embodiment of this aspect, the compound of formula (I) is further 15 characterized by a structure of formula II:

R⁹

II wherein:

Q is C-R¹⁰ or N

R⁹ is CH₃ or CH₂CH₂F; and

R¹⁰ is H or CH₃.

In another embodiment of this aspect, the compound of formula (I) is selected from the group consisting of:

F

In another embodiment of this aspect, the brain cancer is a metastatic brain cancer.

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

Other aspects or embodiments of the présent invention include those as substantially shown and described and any possible combinations of any two or more embodiments described herein.

The terms in the présent application, if not specifically defined, take their ordinary meanings as would be understood by those skilled in the art.

As used herein, the term “halo” or “halogen” refers to F, Cl, or Br.

The term lower alkyl refers to a branched or straight-chain alkyl group having from one to seven carbon atoms, preferably one to four, and more preferably one to two carbon atoms.

The term lower alkoxy refers to an alkoxy group (-OR) having from one to seven, preferably one to four, and more preferably one to two carbon atoms.

The term “cyano” refers to -CN.

The term “pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergie response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.

As used herein, the term pharmaceutically acceptable sait refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animais without undue toxicity, irritation, allergie response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group 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. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycérophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stéarate, succinate, sulfate, tartrate, thiocyanate, //-toluenesulfonate, undecanoate, valerate salts, and the like.

The term “solvaté,” as used herein, means a physical association of a compound of this invention with a stoichiometric or non-stoichiometric amount of solvent molécules. For example, one molécule of the compound associâtes with one or more, preferably one to three, solvent molécules. It is also possible that multiple (e.g., two) molécules of the compound share one solvent molécule. This physical association may include hydrogen bonding. In certain instances the solvatés will be capable of isolation as crystalline solid. The solvent molécules in the solvaté may be présent in a regular arrangement and/or a non-ordered arrangement. Exemplary solvatés include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are generally known in the art.

The term “prodrug,” as used herein, refers to a dérivative of a compound that can be transformed in vivo to yield the parent compound, for example, by hydrolysis in blood. Common examples include, but are not limited to, ester and amide forms of an active carboxylic acid compound; or vice versa, an ester from of an active alcohol compound or an amide form of an active amine compound. Such amide or ester prodrug compounds may be prepared according to conventional methods as known in the art. For example, a prodrug of a compound of formula II of the présent invention could be in the form of the following formula III:

N N y

R^x OCH₃ wherein R^x and R^y are independently H and -C(O)-R, wherein R is C1-C4 alkyl, preferably methyl or ethyl, and more preferably methyl. Other prodrugs of the présent invention can be prepared similarly.

When it is possible that, for use in therapy, therapeutically effective amounts of a compound of the présent invention, or pharmaceutically acceptable salts or solvatés thereof, may be administered as the raw Chemical, it is possible to présent the active ingrédient as a pharmaceutical composition. Accordingly, the disclosure further provides pharmaceutical compositions, which include any compounds of the présent invention, or pharmaceutically acceptable salts or solvatés thereof, and one or more, preferably one to three, pharmaceutically acceptable carriers, diluents, or other excipients. The carrier(s), diluent(s), or other excipient(s) must be acceptable in the sense of being compatible with the other ingrédients of the formulation and not deleterious to the subject being treated.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingrédient per unit dose. Typically, the pharmaceutical compositions of this disclosure will be administered from about 1 to about 5 times per day or altematively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingrédient that may be combined with the carrier materials to produce a single dosage form will vary depending on the condition being treated, the severity of the condition, the time of administration, the route of administration, the rate of excrétion of the compound employed, the duration of treatment, and the âge, gender, weight, and condition of the patient. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingrédient. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the compound. Thereafter, the dosage is increased by small incréments until the optimum effect under the circumstances is reached. In general, the compound is most desirably administered at a concentration level that will generally afford effective results without causing substantial harmful or deleterious side effects.

When the compositions of this disclosure comprise a combination of a compound of the présent disclosure and one or more, preferably one or two, additional therapeutic or prophylactic agent, both the compound and the additional agent are usually présent at dosage levels of between about 10 to 150%, and more preferably between about 10 and 80% of the dosage normally administered in a monotherapy regimen.

Pharmaceutical formulations may be adapted for administration by any appropriate route, for example, by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parentéral (including subcutaneous, intracutaneous, intramuscular, intra-articular, intrasynovial, intrastemal, intrathecal, intralesional, intravenous, or intradermal injections or infusions) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingrédient with the carrier(s) or excipient(s). Oral administration or administration by injection is preferred.

Pharmaceutical formulations adapted for oral administration may be presented as discrète units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid émulsions or water-in-oil émulsions.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as éthanol, glycerol, water, and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agent can also be présent.

Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloïdal silica, talc, magnésium stéarate, calcium stéarate, or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the médicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can 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 acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, and the like. Lubricants used in these dosage forms include sodium oleate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, betonite, xanthan gum, and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture is prepared by mixing the compound, suitable comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelating, or polyvinyl pyrrolidone, a solution retardant such as paraffm, a résorption accelerator such as a quatemary sait and/or and absorption agent such as betonite, kaolin, or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the resuit is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stéarate sait, talc, or minerai oil. The lubricated mixture is then compressed into tablets. The compounds of the présent disclosure can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material, and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups, and élixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while élixirs are prepared through the use of a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners, or saccharin or other artificial sweeteners, and the like can also be added.

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

It should be understood that in addition to the ingrédients 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.

The term patient or “subject” includes both human and other mammals.

The term mammal or “mammalian animal” includes, but is not limited to, humans, dogs, cats, horses, pigs, cows, monkeys, rabbits and mice. The preferred mammals are humans.

The term “therapeutically effective amount refers to an amount of a compound or composition that, when administered to a subject for treating a disease, is suffi ci ent to effect such treatment for the disease. A therapeutically effective amount can vary depending on, inter alia, the compound, the disease and its severity, and the âge, weight, or other factors of the subject to be treated. When applied to an individual active ingrédient, administered alone, the term refers to that ingrédient alone. When applied to a combination, the term refers to combined amounts of the active ingrédients that resuit in the therapeutic effect, whether administered in combination, serially, or simultaneously.

The term treating or “treatment” refers to: (i) inhibiting the disease, disorder, or condition, i.e., arresting its development; (ii) relieving the disease, disorder, or condition, i.e., causing régression of the disease, disorder, and/or condition; or (iii) preventing a disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder, and/or condition but has not yet been diagnosed as having it. Thus, in one embodiment, “treating or treatment refers to ameliorating a disease or disorder, which may include ameliorating one or more physical parameters, though maybe indiscemible by the subject being treated. In another embodiment, treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discemible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, treating or treatment includes delaying the onset of the disease or disorder.

When the term “about” is applied to a parameter, such as amount, température, time, or the like, it indicates that the parameter can usually vary by ±10%, preferably within ±5%, and more preferably within ±2%. As would be understood by a person skilled in the art, when a parameter is not critical, a number provided in the Examples is often given only for illustration purpose, instead of being limiting.

The term “a,” “an,” or “the,” as used herein, represents both singular and plural forms. In general, when either a singular or a plural form of a noun is used, it dénotés both singular and plural forms of the noun.

The following non-limiting Examples further illustrate certain aspects of the présent invention.

EXAMPLES

Materials and Methods

W(2-(2-(Dimcthylamino)ethoxy)-4-methoxy-5-((4-(l-methyl-lH-mdol-3yl)pyrimidin-2-yl)amino)phenyl)acrylamide methanesulfonate (1· Ms; C27H30N₆O3-1.06 CH3SO3H, MW = 486.58, FW = 588.45, purity = 100%) was formulated in a vehicle of 1% polysorbate 80 in water. The complété vehicle was added to the pre-weighed compound to achieve a 5 mg/mL stock solution, suitable for treatment of the 50 mg/kg dose level. The mixture was vortexed for approximately one minute resulting in a light yellow solution with a pH value of 7. The dosing solution was prepared fresh daily.

Animais

Female Envigo Nude mice (Hsd:Athymic ^Mà.e-Foxnl^nu') were used. They were 6-7 weeks old on Day 1 of the experiment. The animais were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum. Animais were housed in static cages with Bed-O’Cobs™ bedding inside Biobubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complété air changes per hour. Ail treatments, body weight déterminations, and tumor measurements were carried out in the bubble environment. The environment was controlled to a température range of 70 ± 2 °F and a humidity range of 3070%.

Cell préparation

NCI-H1975-Luc cells were obtained from Clovis. They were grown in RPMI 1640 medium which was modified with 1 mM Na pyruvate + 2 mM L-glutamine + lOmM HEPES + 2.5 g/L glucose + 5 ug/mL blasticidin and supplemented with 10% non-heatinactivated Fêtai Bovine Sérum (FBS) and 1% 100X Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO2 atmosphère at 37 °C. When expansion was complété, the cells (passage 4) were trypsinized using 0.25% trypsin-EDTA solution. Following cell detachment, the trypsin was inactivated by dilution with complété growth medium and any clumps of cells were separated by pipetting. The cells were centrifuged at 200 rcf for 8 minutes at 4 °C, the supernatant was aspirated, and the pellet was re-suspended in cold Dulbecco's Phosphate Buftered Saline (DPBS) by pipetting. An aliquot of the homogeneous cell suspension was diluted in a trypan blue solution and counted using a Luna automated cell counter to détermine a pre-implantation cell viability. The cell suspension was centrifuged at 200 rcf for 8 minutes at 4°C. The supernatant was aspirated and the cell pellet was re-suspended in cold serum-free medium to generate a final concentration of 1.00E+08 trypanexcluding cells/mL. The cell suspension was maintained on wet ice during implantation. Following implantation, an aliquot of the remaining cells was diluted with a trypan blue solution and counted to détermine the post-implantation cell viability.

Intracranial implantation

Test mice were implanted intracranially on Day 0 with 1.00E+06 cells per 10 pL as per the protocol (Appendix 1). For aseptie surgical implantation, mice were injected with 0.2 mg/kg buprénorphine and anesthetized using 2% isoflurane in air. The mice were then secured in a stereotaxic frame (ASI instruments, Inc.) using non-rupture ear bars. Ocular ointment was applied to the eyes of the mice to prevent drying during surgery. A recirculating 37 °C water heated pad was used to maintain the animal’s body température during the implantation procedure. Once in the stereotaxic frame, the cranium was swabbed with altemating chlorhexidine solution and 70% ethanol-saturated swabs to disinfect the skin surface and préparé for the incision. A 1 cm longitudinal incision was made centrally over bregma of the cranium using a #15 BD scalpel blade. The incision was retracted using small, serrated serrefines. The thin layer of connective tissue covering the surface of the skull was removed using dry cotton swabs under light pressure. Bleeding vessels were cauterized to prevent blood loss. A 0.9 mm drill bit was then centered over bregma, moved 2 mm right latéral, 1 mm anterior to the coronal suture and lowered to score the surface of the skull using the stereotaxic electrode manipulator arm. The drill was removed from the stereotaxic frame and the burr hole through the skull to the surface of the dura mater was completed by hand. The cell suspension (stored on wet ice) was mixed thoroughly and drawn up into a 50 pL gas-tight Hamilton syringe. A standard 27g needle was fîlled with the cell suspension to eliminate air pockets and the luer tip of the syringe was inserted into the needle hub. The syringe was secured to a custom-built syringe holder (ASI Instruments, Inc.) and attached to the stereotaxic frame manipulator arm. The syringe needle was centered over the burr hole and lowered until the beveled tip was level with the underside of the skull at the surface of the dura mater. The needle was then lowered 3mm into the brain and retracted 1mm to form a “réservoir” for the déposition of the cell suspension. 10 pL of the cell suspension (1x10^ cells/mouse) was then injected slowly into the brain tissue with any slight leakage (typical for IC implants) being absorbed with a dry cotton swab. Following the injection, the needle was withdrawn and the burr hole was immediately sealed with bone wax to minimize the loss of implanted cells. The skull surface was then cleaned with altemating dry and 70% éthanol saturated cotton swabs to remove extraneous cells and deter extracranial tumor growth. The mouse was removed from the stereotaxic frame and the incision was closed using a stainless steel wound clip. Once the mouse regained consciousness and dorsal recumbancy, it was retumed to its caging.

Treatment

Ail mice were sorted into treatment groups based on estimation of tumor burden via bioluminescence imaging. The mice were distributed to ensure that the mean tumor burden for ail groups was within 10% of the overall mean tumor burden for the study population. Treatment began on Day 5.

Group 1: Vehicle Control (1% polysorbate 80), 0.2mL/20g, PO, QDxl 1 (once daily on Days 5-15).

Group 2: FMs, 50mg/kg, PO, QDxl4 (once daily on Days 5-18).

Bioluminescence Imaging

In vivo bioluminescence imaging was performed using an IVIS 50 optical imaging (Xenogen, Alameda, CA). Animais were imaged three at a time under ~l-2% isoflurane gas anesthésia. Each mouse was injected IP with l50mg/kg D-luciferin and imaged in the prone position, 10 minutes after the injection. Large binning of the CCD chip was used, and the exposure time was adjusted (2 seconds to 2 minutes) to obtain at least several hundred counts from the metastatic tumors that were observable in each mouse in the image and to avoid saturation of the CCD chip. BLI images were collected on Day 5, 8, 11, 13, 15, and 18. Images were analyzed using the Living Image version 4.3.1 (Xenogen, Alameda, CA) software. Fixed-volume ROIs were placed to encompass the primary tumor on prone images for each individual animal, and labeled based on animal identification. Total flux (photons/sec) was calculated and exported for ail ROIs to facilitate analyses between groups.

Measurements and endpoints %T/C of primary tumor burden (as estimated by BLI) was used as the primary endpoint in this study. %T/C is defined as the médian BLI signal of the treated group divided by the médian BLI signal of the control group x 100. Day 13 %T/C was used for analysis because it was the last day imaging was performed and more than the médian number of animais in the control group remained on study. Life span extension was used as a secondary endpoint in this study. A complété response (CR) is defined as a decrease in tumor mass (based on bioluminescence imaging) to an unreliable signal (below 2.0E+05 photons/sec). Background levels for BLI are typically in the range of 1E+03-1E+04 photons/sec. A partial response (PR) is defined as a > 50% decrease in tumor BLI signal from that at first treatment. PRs are exclusive of CRs.

Efficacy Results

Group 2: LMs, 50mg/kg, PO, QDxl4 (once daily on Days 5-18).

Treatment with PMs produced significant (P<0.05) anticancer activity based on BLI derived Day 13 %T/C (1%; Fig. 1). The médian lifespan was 29.0 days (107% ILS or 15 day increase in lifespan) (p<0.001). By Day 18, 100% of animais had partial tumor régressions, and none had complété régressions (Fig. 2).

Compound 1 also effectively inhibits the kinase domain of the T790M double mutant in addition to the activating mutations and therefore overcomes the résistance observed with the currently used therapy of réversible inhibitors. Since the rôle of EGFR in non-small cell lung cancer (NSCLC) is well-established (Ohashi, K.; et al. J. Clin. Oncol. 2013, 31, 1070), 1 represents a potential therapeutic agent for the treatment of non-small cell lung cancer.

Compound 1 achieves therapeutic levels of brain concentration when dosed orally in the rat (Table 1). Furthermore, 1 was efficacious against intracranially implanted brain 5 tumors in mice. Therefore, 1 represents a potential therapeutic agent for the treatment of EGFR-mediated metastatic brain cancer.

Treatment with 1-Ms was well-tolerated and produced significant (P<0.05) anticancer activity based on Day 13 BLI %T/C and lifespan. Although ail mice eventually died of disease, life span was more than doubled by treatment with 1-Ms.

Table 1. Pharmacokinetic (PK) parameters for 1-Ms in male SD rats dosed PO @ 5 mg/kg using a 0.5% methylcellulose vehicle.

Parameter	Plasma	Brain
Cmax (ng/mL or ng/g)	96.3	606
Tmax (h)	2.00	2.00
T1/2(h)	1.97	2.01
Tlast (h)	18.0	18.0
AUCo-iast (ng-h/mL or ng-h/g)	535	5047
AUCo-inf (ng-h/mL or ng-h/g)	538	5085
MRTo-iast (h)	5.01	5.95
MRT0-inf(h)	5.10	6.07
AUCExtra(%)	0.593	0.746
AUMCExtra(%)	2.42	2.57
dAUC Ratio	—	9.44

The foregoing examples or preferred embodiments are provided for illustration 15 purpose and are not intended to limit the présent invention. Numerous variations and combinations of the features set forth above can be utilized without departing from the présent invention as set forth in the claims.

Claims (10)

1. Use of a compound of formula (I):

(I) or a pharmaceutically acceptable sait, solvaté, prodrug, or composition thereof, in the manufacture of a médicament for the treatment of brain cancer, wherein:

R¹ is selected from hydrogen, halogen, methyl, trifluoromethyl, and cyano;

R², R³, and R⁴ are the same or different and are independently selected from hydrogen, halogen, and trifluoromethyl;

R⁵ is selected from lower alkyl, optionally substituted 3- to 6-membered heterocyclyl, R⁷R⁸N(lower alkyl), and R⁷R⁸N-(cycloalkylalkyl), wherein R⁷ and R⁸ are the same or different and are independently selected from hydrogen and lower alkyl;

R⁶ is selected from lower alkoxy and lower alkyl; and

Qis C-R¹⁰orN

R⁹ is C_rC₄ alkyl or C1-C4 haloalkyl; and

R¹⁰ is H or CH₃.

2. The use of claim 1, wherein:

R¹ is hydrogen or halogen, or methyl;

R² is hydrogen or halogen; and

R⁵ is selected from methyl, R⁷R⁸N-(CH₂)_n- (n = 2 or 3), 1 -(dimethylamino)cyclopropylmethyl, 3-(dimethylamino)cyclobutyl, l-methylazetidin-3-yl, (/?)-!methylpyrrolidin-3-yl, (ô)-l-methylpyrrolidin-3-yl, and l-methylpiperidin-4-yl.

3. The use of claim 1 or 2, wherein:

R² is hydrogen, F, or Cl;

R³ is hydrogen, F, Cl, or -CF₃;

R⁴ is hydrogen; and

R⁵ is 2-dimethylamino-ethyl [(CH₃)₂NCH₂CH₂-].

4. The use of any one of claims 1 to 3, wherein R¹, R², R³, and R⁴ are ail hydrogen.

5. The use of claim 1, wherein the compound of formula (I) is further characterized by a structure of formula II:

II wherein:

Q is C-R¹⁰ or N

R⁹ is CH₃ or CH₂CH₂F; and

R¹⁰ is H or CH₃.

6. The use of claim 5, wherein the compound of formula (I) is further characterized by a structure of formula:

N

7. The use of claim 1, wherein the compound of formula (I) is selected from the group consisting of:

N—

O.

N H

N H

N H

N ^H O

5

8. The use of any one of claims 1 to 7, wherein said brain cancer is a metastatic brain cancer.

9. The use of any one of claims 1 to 7, wherein said brain cancer is metastatic brain cancer developed from an EGFR-mediated non-small cell lung cancer.

10. The use of any one of claims 1 to 9, wherein said compound of formula (I) or (II) is

10 the methanesulfonic acid sait of compound 1 (1-Ms).