CN108912095A

CN108912095A - Benzimidazoles compound and its preparation method and application

Info

Publication number: CN108912095A
Application number: CN201810901513.8A
Authority: CN
Inventors: 谢德逊; 李权伟; 薛伟才; 刘辉
Original assignee: Guangzhou Iwahito Medical Technology Co Ltd
Current assignee: Guangzhou Iwahito Medical Technology Co Ltd
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2018-11-30
Anticipated expiration: 2038-08-09
Also published as: CN108912095B

Abstract

The present invention relates to a kind of benzimidazoles compound and its applications.For the structure of the benzimidazoles compound as shown in I, which shows preferable FAK inhibitory activity as focal adhesion kinase inhibitor.At the same time, have stronger drug effect, better medicine for property and/or toxicological characteristics, such as：Good brain/blood plasma than, good bioavilability, good metabolic stability and its reduce the inhibition that acts on mitochondrial respiratory, have preferable potential applicability in clinical practice.

Description

Benzimidazole compound and preparation method and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to benzimidazole compounds, and a preparation method and application thereof.

Background

Focal Adhesion Kinase (FAK) was discovered in 1992 as an intracellular non-receptor tyrosine kinase that is expressed in cells of various tissues and has a regulatory role in many cells. FAK is an early regulator of integrin signaling cascade, so that integrin clustering in response to various stimuli produces FAK autophosphorylation at Tyr397, activates a large number of downstream signaling pathways, and regulates biological behaviors of cells such as migration, proliferation, differentiation, and the like. In normal cells, FAK regulates a variety of essential cellular functions such as proliferation and growth, prevention of apoptosis, adhesion, and cell spreading, invasion, and migration. Within cancer cells, FAK is the junction of multiple signaling pathways. Abnormally overexpressed FAK has been detected in a large number of human cancer cells, such as breast cancer, ovarian/colon cancer, prostate cancer, colon cancer, and the like. Based on the important role of FAK in tumorigenesis, migration, proliferation and apoptosis, the FAK inhibitor can inhibit the activity of FAK and block the rapid proliferation and diffusion of cancer cells, so that the FAK inhibitor is designed and synthesized to have important significance for treating cancers.

Furthermore, in more intensive studies and studies on mouse models in which FAK was knocked out, it was shown that: FAK plays a key role in the formation of blood vessels during embryonic growth and cancer cell growth. FAK is therefore useful in the treatment of pathological angiogenesis, for example as an anti-angiogenic therapy in diseases such as cancer and retinopathy.

Based on the above two points, FAK is one of the currently widely focused anti-tumor gold targets, and FKA inhibitor has become a hot spot for research of various large pharmaceutical companies in recent years.

Disclosure of Invention

Based on the above, the invention provides a novel benzimidazole compound which is used as an inhibitor of focal adhesion kinase and shows better FAK inhibitory activity.

The specific technical scheme is as follows:

a benzimidazole compound having a structure represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite, prodrug, or mixture thereof:

wherein,

R^Nselected from hydrogen, methyl, isopropyl;

R^Cselected from hydrogen, methoxy, fluorine.

In one embodiment, R is^NSelected from methyl, isopropyl;

R^Cselected from hydrogen, methoxy, fluorine.

In one embodiment, the benzimidazole compound has the structure of formula (II):

wherein,

R^Nselected from methyl, isopropyl;

R^Cselected from hydrogen and fluorine.

In one embodiment, the benzimidazole compound has the structure of formula (III):

wherein,

R^Nis selected from methyl;

R^Cselected from methoxy and fluorine.

In one embodiment, the benzimidazole compound has one of the following structures:

in one embodiment, the benzimidazole compound is selected from the group consisting of:

2- ((2- ((1H-benzo [ d ] imidazol-2-yl) amino) -5-chloropyridin-4-yl) amino) -N-methoxybenzamide,

2- ((5-chloro-2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide,

2- ((5-chloro-2- ((6-methoxy-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide,

2- ((5-chloro-2- ((6-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide,

2- ((5-chloro-2- ((7-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide,

2- ((5-chloro-2- ((1-isopropyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide.

The invention also provides a medicament.

The specific technical scheme is as follows:

a medicament comprising the benzimidazole compound or pharmaceutically acceptable salts, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, prodrugs or mixtures thereof, and pharmaceutically acceptable excipients.

In one embodiment, the active ingredient of the medicament further comprises an additional therapeutic agent.

In one embodiment, the additional therapeutic agent comprises one or more of an anti-cancer agent and a drug for treating pulmonary hypertension.

The invention also provides application of the compound in preparing a medicament for preventing and treating FAK related diseases.

In one embodiment, the FAK-related disease comprises: cancer, pulmonary hypertension, or pathological angiogenesis.

The invention provides a novel benzimidazole compound which is used as an inhibitor of focal adhesion kinase and shows good FAK inhibitory activity. Meanwhile, the compound has stronger drug effect, better pharmacokinetic property and/or toxicological property, such as: good brain/plasma ratio, good bioavailability, good metabolic stability, and can reduce inhibition on mitochondrial respiration, and has good clinical application prospect.

Detailed Description

Description of the benzimidazole Compounds of the invention

The invention provides benzimidazole compounds with a structure shown in a formula (I) or pharmaceutically acceptable salts, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, prodrugs or mixtures thereof:

wherein,

R^Nselected from hydrogen, methyl, isopropyl;

R^Cselected from hydrogen, methoxy, fluorine.

Unless otherwise indicated, all stereoisomers, geometric isomers, tautomers, nitroxides, solvates, metabolites, salts and pharmaceutically acceptable prodrugs of the compounds of the invention are within the scope of the present invention.

In particular, the salts are pharmaceutically acceptable salts. The term "pharmaceutically acceptable" includes substances or compositions, which must be compatible with chemical or toxicology, and which are related to the other components making up the formulation and the mammal being treated.

Salts of the compounds of the present invention also include, but are not necessarily pharmaceutically acceptable salts of intermediates used in the preparation or purification of the benzimidazoles of formula (I) or isolated enantiomers of the benzimidazoles of formula (I).

Unless otherwise indicated, the structural formulae depicted herein include all isomeric forms (e.g., enantiomeric, diastereomeric, and geometric (or conformational) isomers): such as the R, S configuration containing an asymmetric center, the (Z), (E) isomers of the double bond, and the conformational isomers of (Z), (E). Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers, or geometric isomers (or conformers) thereof are within the scope of the present invention.

Unless otherwise indicated, all tautomeric forms of the compounds of the invention are included within the scope of the invention. In addition, unless otherwise indicated, the structural formulae of the compounds described herein include isotopically enriched concentrations of one or more different atoms.

The compounds of the present disclosure may contain asymmetric or chiral centers and thus may exist in different stereoisomeric forms. The present invention is directed to all stereoisomeric forms of the compounds of formula (I) benzimidazoles, including but not limited to diastereomers, enantiomers, atropisomers and geometric (or conformational) isomers, and mixtures thereof, such as racemic mixtures, as part of the present invention.

In the structures disclosed herein, when the stereochemistry of any particular chiral atom is not specified, then all stereoisomers of that structure are contemplated as within this invention and are included as disclosed compounds in this invention. When stereochemistry is indicated by a solid wedge (solid wedge) or dashed line representing a particular configuration, then the stereoisomers of the structure are so well-defined and defined.

Nitroxides of the compounds of the present invention are also included within the scope of the present invention. The nitroxides of the compounds of the present invention may be prepared by oxidation of the corresponding nitrogen-containing basic species using a common oxidizing agent (e.g. hydrogen peroxide) in the presence of an acid such as acetic acid at elevated temperature, or by reaction with a peracid in a suitable solvent, for example peracetic acid in dichloromethane, ethyl acetate or methyl acetate, or 3-chloroperoxybenzoic acid in chloroform or dichloromethane.

If the compounds of the invention are basic, the desired salts may be prepared by any suitable method provided in the literature, for example, using inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids and the like, or using organic acids such as acetic, maleic, succinic, mandelic, fumaric, malonic, pyruvic, oxalic, glycolic and salicylic acids, pyranonic acids such as glucuronic and galacturonic acids, α -hydroxy acids such as citric and tartaric acids, amino acids such as aspartic and glutamic acids, aromatic acids such as benzoic and cinnamic acids, sulfonic acids such as p-toluenesulfonic, ethanesulfonic, and the like.

If the compounds of the invention are acidic, the desired salts can be prepared by suitable methods, e.g., using inorganic or organic bases, such as ammonia (primary, secondary, tertiary), alkali or alkaline earth metal hydroxides, and the like. Suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia such as primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine, piperazine and the like, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

Benzimidazole compound, pharmaceutical composition, preparation and administration thereof

The pharmaceutical composition of the present invention is characterized by comprising the benzimidazole compound represented by the formula (I), the compounds listed in the present invention, or the compounds of the examples. The amount of compound in the compositions of the invention is effective to treat or ameliorate a FAK kinase-associated disorder in a patient.

As described herein, the pharmaceutically acceptable compositions of the present invention further comprise pharmaceutically acceptable excipients, which as used herein, include any solvent, diluent, or other liquid excipient, dispersing or suspending agent, surfactant, isotonic agent, thickening agent, emulsifier, preservative, solid binder or lubricant, and the like, as appropriate for the particular target dosage form. As described in the following documents: in Remington, The Science and Practice of pharmacy,21st edition,2005, ed.D.B.Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds.J.Swarbrickand J.C.Boylan, 1988. Annu 1999, Marcel Dekker, New York, taken together with The disclosure of this document, suggests that different excipients may be used In The formulation of pharmaceutically acceptable compositions and their well known methods of preparation. Except to the extent that any conventional excipient is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention.

The pharmaceutical compositions of the invention further comprise i) one or more other FAK inhibitors and/or ii) one or more other types of protein kinase inhibitors and/or one or more other types of therapeutic agents. Wherein the one or more other types of protein kinase inhibitors include, for example, PYK2 or src inhibitors, and the other types of therapeutic agents include other anti-cancer agents, other agents for treating pulmonary hypertension, and the like.

When useful therapeutically, a therapeutically effective amount of the benzimidazole compounds of the invention, particularly the benzimidazole compounds of formula (I) and pharmaceutically acceptable salts thereof, may be administered as raw chemicals and may be provided as the active ingredient of a pharmaceutical composition. Accordingly, the present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention, particularly a benzimidazole compound of formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients. The term "therapeutically effective amount" as used herein refers to the total amount of each active component sufficient to show meaningful patient benefit (e.g., reduction in viral load). When the active ingredient alone is used for separate administration, the term refers only to that ingredient. When used in combination, the term refers to the combined amounts of the active ingredients that, when combined, administered sequentially or simultaneously, result in a therapeutic effect. The carrier, diluent or excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. According to a further aspect of the present disclosure there is also provided a process for the preparation of a pharmaceutical formulation which comprises mixing a compound of the present invention, especially a benzimidazole of formula (I) or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients. The term "pharmaceutically acceptable" as used herein refers to 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, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use.

The amount of active ingredient combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, formulations intended for oral administration to humans will typically comprise, for example, 0.5mg-2g of the active ingredient (suitably 0.5mg-1g of the active ingredient, for example 0.5mg-0.5g of the active agent, more suitably 0.5-100mg, for example 1-30mg) complexed with a suitable and convenient amount of excipient which may be from about 5% to about 98% by weight of the total composition. The amount of active ingredient which is combined with the compound of formula (I) and the carrier material to prepare a single dosage form will vary depending upon the disease to be treated, the severity of the disease, the time of administration, the route of administration, the rate of excretion of the compound employed, the time of treatment and the age, sex, body weight and condition of the patient. Preferred unit dosage forms are those containing a daily or divided dose or suitable fraction thereof of the active ingredient described herein above. Treatment can be initiated with small doses, which are clearly below the optimal dose of the compound. Thereafter, the dosage is increased in smaller increments until the optimum effect is achieved in this case. In general, the compounds are most desirably administered at concentration levels that generally provide effective results in antitumor terms without causing any harmful or toxic side effects.

In the course of therapy or prophylaxis with the compounds of the invention, if divided doses are required, they will generally be administered so that a daily dose in the range 0.1mg/kg to 75mg/kg is obtained. Lower doses will be administered when the parenteral route is employed. For example, for intravenous or intraperitoneal administration, a dosage of 0.1mg/kg to 30mg/kg is generally used. Also, for administration by inhalation, a dose of 0.05mg/kg to 25mg/kg will be employed. Oral administration is also suitable, particularly in the form of tablets. Typically, a unit dosage form will contain from about 0.5mg to 0.5g of a compound of the invention and the unit dosage form may be administered once, twice, three or four times daily or, if desired, more frequently.

Pharmaceutical dosage forms of the compounds of the invention and compositions thereof may be provided in the form of immediate release, controlled release, sustained release or targeted drug release systems. For example, common dosage forms include solutions and suspensions, (micro) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols and freeze-dried preparations. Depending on the route of administration used, Special devices may be required to administer or administer the drug, such as syringes and needles, inhalers, pumps, injection pens, applicators or Special bottles (Special flash). Pharmaceutical dosage forms often consist of a drug, excipients, and a container/closure system. One or more excipients (also known as inactive ingredients) may be added to the compounds of the present invention to improve or facilitate the manufacture, stability, administration, and safety of the drug, and may provide a means to obtain the desired drug release profile. Thus, the type of excipient added to a drug may depend on various factors, such as the physical and chemical properties of the drug, the route of administration, and the preparation steps. Pharmaceutically acceptable excipients exist in the art and include those listed in various pharmacopoeias. (see U.S. Pharmacopoeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP) and British Pharmacopoeia (BP)), the United states food and Drug Administration (the U.S. food and Drug Administration, www.fda.gov) Drug Evaluation and Research Center (CEDR) publications, such as "Guide for inactive ingredients" (Ingredient Guide,1996), Ash and Ash written "Handbook of pharmaceutical Additives (2002, United states Information Resources, Inc.; ethics).

The pharmaceutical compositions are suitable for administration by any suitable route, for example by the 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 or subdermal injection or infusion) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with a carrier or excipient. Oral administration or injection administration is preferred.

Pharmaceutical formulations adapted for oral administration are provided in discrete units, such as capsules or tablets; powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foam or foam formulations (whip); or an oil-in-water emulsion or a water-in-oil emulsion.

For example, for oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient may be mixed with a pharmaceutically acceptable oral, non-toxic inert carrier (e.g., ethanol, glycerol, water, etc.). Powders are prepared by pulverizing the compound to a suitable fine size and mixing with a pharmaceutically acceptable carrier (e.g., an edible sugar such as starch or mannitol) which is also pulverized. Flavoring, preservative, dispersing and coloring agents may also be present.

Capsules are prepared by preparing a powdered mixture as described above and filling into shaped gelatin shells. Glidants and lubricants (e.g., colloidal silicon dioxide, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol) may be added to the powder mixture prior to the filling operation. Disintegrating or solubilizing agents (e.g., agar-agar, calcium carbonate or sodium carbonate) that will improve the availability of the drug when the capsule is taken can also be added.

Suitable binders include starch, gelatin, natural sugars (e.g., glucose or β -lactose), corn sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol, and the like, lubricants for such 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.

Oral liquid preparations such as solutions, syrups and elixirs 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 elixirs can be prepared through the use of non-toxic vehicles. Solubilizing agents and emulsifiers (e.g., ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers), preservatives, flavoring additives (e.g., peppermint oil or natural sweeteners or saccharin or other artificial sweeteners), and the like may also be added.

Dosage unit formulations for oral administration may be microencapsulated, if appropriate. The formulations may also be formulated for extended or sustained release, for example by coating or embedding in a particulate material such as a polymer, wax or the like.

The compounds of the present invention, particularly the benzimidazoles of formula (I) and pharmaceutically acceptable salts thereof, may also be administered in liposomal delivery systems, such as small unilamellar liposomes, large unilamellar liposomes, and multilamellar liposomes. Liposomes can be composed of a variety of phospholipids (e.g., cholesterol, octadecylamine, or phosphatidylcholine).

The compounds of the present invention, particularly the benzimidazoles of formula (I) and pharmaceutically acceptable salts thereof, may also be delivered by using monoclonal antibodies as the sole carrier to which the compound molecules are coupled. The compounds may also be conjugated to soluble polymers as targetable drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide phenol, polyhydroxyethylaspartamide phenol, or polyethyleneoxide polylysine substituted with palmitoyl residues. In addition, the compounds may be coupled to a class of biodegradable polymers for achieving controlled release of a drug, such as polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches (patches) to remain in intimate contact with the epidermis of the recipient for an extended period of time. For example, the active ingredient may be delivered by iontophoretic patches, as generally described in Pharmaceutical Research 1986,3(6), 318.

Pharmaceutical preparations suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils or transdermal patches.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed amkside and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. The injection solution and suspension can be prepared into sterile powder for injection, granule and tablet.

Pharmaceutical formulations adapted for nasal administration (wherein the carrier is a solid) comprising a coarse powder having a particle size in the range 20 to 500 microns are administered by nasal inhalation, i.e. by rapid inhalation through the nasal passage from a container of the coarse powder close to the nose. Suitable formulations in which the carrier is a liquid, suitable for administration as a nasal spray or nasal drops, include aqueous or oily solutions of the active ingredient.

Pharmaceutical formulations suitable for administration by inhalation include dry powder, aerosol, suspension or solution compositions.

Dry powder compositions for delivery to the lung by inhalation typically comprise a benzimidazole compound of formula (I) or a pharmaceutically acceptable salt of the invention as a finely divided powder, together with one or more pharmaceutically acceptable excipients as a finely divided powder. Pharmaceutically acceptable excipients that are particularly suitable for use in dry powders are known to those skilled in the art and include lactose, starch, mannitol and mono-, di-and polysaccharides. Finely divided powders can be prepared, for example, by micronization (micronisation) and milling. In general, a size-reduced (e.g., micronized) compound can be defined by a D50 value (e.g., as detected using laser diffraction) of about 1 to about 10 microns.

The dry powder can be administered to a patient via a Reservoir Dry Powder Inhaler (RDPI) having a reservoir adapted to store multiple administrations (unmetered doses) of the drug in dry powder form. RDPI typically includes a device for metering each dose of drug from the reservoir to the delivery site. For example, the metering device may comprise a metering cup that is movable from a first position, in which the cup can be loaded with medicament from a reservoir, to a second position, in which a metered dose of medicament is prepared for inhalation by a patient.

Alternatively, the dry powder may be presented as a capsule (such as gelatin or plastic), cartridge, or blister pack for a multi-dose dry powder inhaler (MDPI). MDPI is an inhaler in which the medicament is contained in a multi-dose package containing (or otherwise carrying) multiple defined doses (or portions thereof) of the medicament. When the dry powder dose is presented as a blister pack, it comprises a plurality of blisters for containing the medicament in dry powder dose form. The blisters are typically arranged in a regular pattern to facilitate release of the medicament therefrom. For example, the blisters may be arranged in a generally circular pattern on a disc-type blister pack, or may be elongated to include, for example, a strip or band form.

An aerosol formulation may be formed by suspending or dissolving the benzimidazole compounds of formula (I) or pharmaceutically acceptable salts thereof of the present invention in a liquefied propellant. Suitable propellants include halogenated hydrocarbons, hydrocarbons and other liquefied gases. Representative propellants include: trichlorofluoromethane (propellant 11), dichlorofluoromethane (propellant 12), dichlorotetrafluoroethane (propellant 114), tetrafluoroethane (HFA-134a), 1-difluoroethane (HFA-152a), difluoromethane (HFA-32), pentafluoroethane (HFA-12), heptafluoropropane (HFA-227a), perfluoropropane, perfluorobutane, perfluoropentane, butane, isobutane and pentane. An aerosol comprising the polymorph or salt of the invention is typically administered to a patient via a Metered Dose Inhaler (MDI). Such devices are known to those skilled in the art.

The aerosol may contain additional pharmaceutically acceptable excipients that are commonly used with MDIs, such as surfactants, lubricants, co-solvents, and other excipients to improve the physical stability of the formulation, improve valve characteristics, improve solubility, or improve taste.

It will be appreciated that in addition to the ingredients particularly mentioned above, the formulations may include other ingredients conventional in the art having regard to the type of formulation in question, for example, such formulations which are suitable for oral administration may include flavouring agents.

Use of the Compounds and pharmaceutical compositions of the invention

Pharmaceutical compositions of the invention may be characterized as comprising a compound of the benzimidazole of formula (I), a compound of the invention as set forth in the examples, or a compound of the examples, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The compounds in the composition of the present invention are useful for the prevention and treatment of FAK-related diseases, including cancer, pulmonary hypertension, immune diseases, arthritis, inflammatory bowel disease, pathological angiogenesis-related diseases, and the like. The compound and the pharmaceutical composition thereof are particularly used for preparing the drugs for treating cancers, pulmonary hypertension and diseases related to pathological angiogenesis. The compounds of the invention or pharmaceutical compositions thereof may be used therapeutically in combination with i) one or more other FAK inhibitors and/or ii) one or more other types of protein kinase inhibitors and/or one or more other types of therapeutic agents, which may be administered orally in the same dosage form, orally in separate oral dosage forms (e.g., sequentially or non-sequentially), or administered together or separately (e.g., sequentially or non-sequentially) by injection. The other types of therapeutic agents include other anticancer agents and other agents for treating pulmonary hypertension.

The compounds of the invention or pharmaceutical compositions thereof are expected to have, among other properties, anti-tumor properties which are believed to result from inhibition of FAK, e.g. the compounds or pharmaceutical compositions thereof may exhibit anti-proliferative and/or pro-apoptotic and/or anti-invasive and/or anti-cell motility and/or anti-angiogenic activity. Such compounds appear to be useful in the treatment of, for example, FAK-induced tumors, particularly as anti-cancer agents.

Accordingly, it is contemplated that the compounds of the present invention, or pharmaceutical compositions thereof, may be useful in the treatment of diseases or medical conditions mediated alone or in part by FAK, i.e., the compounds may be useful in producing a FAK inhibitory effect in a warm-blooded animal in need of such treatment. Thus, the compounds of the present invention provide a method of treating malignant cells characterized by inhibition of FAK. In particular, the compounds of the present invention or pharmaceutical compositions thereof may be used to produce an anti-proliferative and/or pro-apoptotic and/or anti-invasive and/or anti-cell motility and/or anti-angiogenic activity effect mediated alone or in part by inhibition of FAK function. In particular, the compounds of the invention are expected to be useful in the prevention or treatment of those tumors that are sensitive to inhibition of FAK, which tumors are associated with, for example, angiogenesis, proliferation and signal transduction steps that result in the proliferation, invasion, migration and, in particular, angiogenesis of these tumor cells. Accordingly, the compounds of the present invention are useful for the treatment of hyperproliferative diseases, including cancer. Benign or malignant tumors may affect any tissue and include non-solid tumors such as leukemia, multiple myeloma or lymphoma, and solid tumors such as bile duct cancer, bone cancer (including ewing's tumor), bladder cancer, brain/CNS cancer, breast cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, liver cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), neural cancer (including neuroblastoma), esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, testicular cancer, thyroid cancer, uterine cancer, cervical cancer and vulvar cancer, and kaposi's sarcoma. It is contemplated that the compounds of the present invention may be useful in the treatment of pathogenic angiogenesis (pathologic angiogenesis), for example in the treatment of cancer as described hereinabove and other diseases that produce inappropriate or pathogenic angiogenesis, such as age-related macular degeneration (AMD) and cancers associated with solid tumors.

The compounds of the present invention or pharmaceutical compositions thereof can be used for the preparation of a medicament particularly useful for the treatment of cancer, including tumors, such as skin cancer, breast cancer, brain cancer, neck cancer, testicular cancer, and the like. It is particularly useful for the treatment of metastatic or malignant tumors. More particularly, cancers that may be treated by the compositions and methods of the present invention include, but are not limited to, the following tumor types: such as astrocytic, breast, neck, colorectal, endometrial, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, oral, ovarian, prostate and thyroid cancers, as well as sarcomas. More particularly, these compounds are useful in the treatment of: cancer in the heart region: sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; lung cancer: bronchial carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, hamartoma, mesothelioma; cancer in the gastrointestinal tract: esophageal cancer (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastric cancer (carcinoma, lymphoma, leiomyosarcoma), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, reoma), small intestine cancer (adenocarcinoma, lymphoma, carcinoid tumor, kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine cancer (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract cancer: renal cancer (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urinary tract cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); cancer of the liver site: liver cancer (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone-related cancers: osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrosarcoma, chondrosarcoma, ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, malignant giant cell chordoma, osteochondromatoma (osteoectochondromy wart), benign chondroma, chondroblastoma-like fibroma (chondroblastoma), osteoid osteoma, and giant cell tumor; cancers of the nervous system: cranial cancers (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meningeal cancers (meningioma, meningiosarcoma (menigiospora), glioma), brain cancers (astrocytoma, neuroblastoma, glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma); gynecological cancer: uterine cancer (endometrial cancer), cervical cancer (neck cancer, pre-neoplastic cervical dysplasia), ovarian cancer (serous cyst adenocarcinoma, mucinous cyst adenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors (granulosa-thecal tumors), sertoli cell tumors, dysgerminoma, malignant teratoma), vulval cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (rhabdomyosarcoma), fallopian tube tumor (carcinoma), hematologic cancer: leukemia (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), hodgkin's disease, pre-tumor cervical dysplasia, cervical cancer (cervical cancer, pre-malignant cyst-cervical cancer), uterine cancer-cervical cancer (serous cyst adenocarcinoma, mucinous cyst adenocarcinoma, sarcoidosis, dysgerminoma, malignant teratoma), vulval cancer (squamous cell carcinoma, botryoid sarcoma, botryoid, Non-hodgkin lymphoma (malignant lymphoma); skin cancer: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal cancer, neuroblastoma. Thus, the term "cancer cell" as provided herein includes cells having any or related of the above-identified conditions.

The compounds of the present invention or pharmaceutical compositions thereof may be used alone or, if desired, in combination with other active compounds. Compounds having the formula (I) benzimidazoles are considered effective when used together with: alkylating agents, angiogenesis inhibitors, antibodies, metabolic antagonists, antimitotic agents, antiproliferative agents, antiviral agents, aurora kinase inhibitors, other apoptosis-promoting agents (e.g., Bcl-xL, Bcl-w, and Bfl-1) inhibitors, activators of death receptor pathways, Bcr-Abl kinase inhibitors, BiTE (Bi-specific T cell Engager (engage)) antibodies, antibody drug conjugates, biological response modifiers, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, Ds DVs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, Heat Shock Protein (HSP) -90 inhibitors, Histone Deacetylase (HDAC) inhibitors, hormonal therapies, immunity, Inhibitors of Apoptosis Proteins (IAPs), warm-blooded animal targeting with the insertion of antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, rapamycin inhibitors, micrornas, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate) -ribose polymerase (PARP) inhibitors, platinum chemotherapy, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteosome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, ergotamine tartrate (etinoids)/sparganium stodelids (todeids) plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, and the like, and combinations of one or more of these agents.

In some embodiments, when administered in combination, there are two modes: 1) the compound or the pharmaceutical composition and other combinable active medicaments are respectively prepared into separate preparations, and the two preparations can be the same or different and can be used sequentially or simultaneously; when used sequentially, the first medicament does not lose its effective effect in vivo when the second medicament is administered; 2) the compound or pharmaceutical composition of the present invention and other active agents that can be combined are formulated into a single formulation and administered simultaneously.

The benzimidazole compounds of formula (I) or pharmaceutically acceptable salts thereof of the present invention may be administered in combination with other therapeutic agents, including other therapeutic agents useful for the treatment of pulmonary arterial hypertension (PHA). These therapeutic agents include vasodilators, such as epoprostenol (Flolan)^TM) Tadalafil (Adcirca)^TM) Or ambrisentan (Volibris)^TM) And so on.

An "effective amount" or "effective dose" of a compound or pharmaceutically acceptable composition of the invention refers to an amount effective to treat or reduce the severity of one or more of the conditions mentioned herein. The compounds and compositions according to the methods of the present invention can be administered in any amount and by any route effective to treat or reduce the severity of the disease. The exact amount necessary will vary depending on the patient, depending on the race, age, general condition of the patient, severity of infection, particular factors, mode of administration, and the like. The compound or composition may be administered in combination with one or more other therapeutic agents.

Detailed Description

In general, the compounds of the present invention may be prepared by the methods described herein, wherein the substituents are as defined in formula (I), unless otherwise indicated. The following reaction schemes and examples serve to further illustrate the context of the invention.

Those skilled in the art will recognize that: the chemical reactions described herein may be used to suitably prepare a number of other compounds of the invention, and other methods for preparing the compounds of the invention are considered to be within the scope of the invention. For example, the synthesis of those non-exemplified compounds according to the present invention can be successfully accomplished by those skilled in the art by modification, such as appropriate protection of interfering groups, by the use of other known reagents in addition to those described herein, or by some routine modification of reaction conditions. In addition, the reactions disclosed herein or known reaction conditions are also recognized as being applicable to the preparation of other compounds of the present invention.

The examples described below, unless otherwise indicated, are all temperatures set forth in degrees Celsius. Reagents were purchased from commercial suppliers such as Lingkai medicine, Aldrich Chemical Company, Inc., Arco Chemical Company and Alfa Chemical Company, and were used without further purification unless otherwise indicated. General reagents were purchased from Shantou Wen Long chemical reagent factory, Guangdong Guanghua chemical reagent factory, Guangzhou chemical reagent factory, Tianjin HaoLiyu Chemicals Co., Ltd, Qingdao Tenglong chemical reagent Co., Ltd, and Qingdao Kaseiki chemical plant.

The anhydrous tetrahydrofuran is obtained by refluxing and drying the metallic sodium. The anhydrous dichloromethane and chloroform are obtained by calcium hydride reflux drying. Ethyl acetate, N, N-dimethylacetamide and petroleum ether were used dried over anhydrous sodium sulfate in advance.

The following reactions are generally carried out under positive pressure of nitrogen or argon or by sleeving a dry tube over an anhydrous solvent (unless otherwise indicated), the reaction vial being stoppered with a suitable rubber stopper and the substrate being injected by syringe. The glassware was dried.

The column chromatography is performed using a silica gel column. Silica gel (300 and 400 meshes) was purchased from Qingdao oceanic chemical plants. Nuclear magnetic resonance spectroscopy with CDC1₃Or DMSO-d₆As solvent (reported in ppm units)TMS (0ppm) or chloroform (7.25ppm) was used as reference standard. When multiple peaks occur, the following abbreviations will be used: s (singleton), d (doublet), t (triplet ), m (multiplet, multiplet), br (broad ), dd (doublet of doublets, quartet), dt (doublet of triplets). Coupling constants are expressed in hertz (Hz).

Low resolution Mass Spectral (MS) data were measured by an Agilent 6320 series LC-MS spectrometer equipped with a G1312A binary pump and a G1316A TCC (column temperature maintained at 30 ℃), a G1329A autosampler and a G1315B DAD detector were applied for analysis, and an ESI source was applied to the LC-MS spectrometer.

Low resolution Mass Spectral (MS) data were determined by Agilent 6120 series LC-MS spectrometer equipped with a G1311A quaternary pump and a G1316A TCC (column temperature maintained at 30 ℃), a G1329A autosampler and a G1315D DAD detector were used for analysis, and an ESI source was used for the LC-MS spectrometer.

Both spectrometers were equipped with an Agilent Zorbax SB-C18 column, 2.1X 30mm, 5 μm. The injection volume is determined by the sample concentration; the flow rate is 0.6 mL/min; peaks of HPLC were recorded by UV-Vis wavelength at 210nm and 254 nm. The mobile phases were 0.1% formic acid in acetonitrile (phase a) and 0.1% formic acid in ultrapure water (phase B). Gradient elution conditions are shown in table 1:

TABLE 1

Time (min)	A(CH₃CN，0.1％HCOOH)	B(H₂O，0.1％HCOOH)
			0-3	5-100	95-0
3-6	100	0
			6-6.1	100-5	0-95
6.1-8	5	95

Compound purification was assessed by Agilent 1100 series High Performance Liquid Chromatography (HPLC) with UV detection at 210nm and 254nm, a Zorbax SB-C18 column, 2.1X 30mm, 4 μm, 10min, flow rate 0.6mL/min, 5-95% (0.1% formic acid in acetonitrile) in (0.1% formic acid in water), the column temperature was maintained at 40 ℃.

The following acronyms are used throughout the invention:

XantPhos 4, 5-bis diphenylphosphine-9, 9-dimethyl xanthene

Cs₂CO₃Cesium carbonate

Pd2(dba)3,Pd₂(dba)₃Tris (dibenzylideneacetone) dipalladium

Pd(OAc)₂Palladium acetate

HCl hydrochloric acid

PE Petroleum Ether

EA, EtOAc ethyl acetate

DCM dichloromethane

DCE 1, 2-dichloroethane

CDCl₃Deuterated chloroform

CH₃OH, MeOH methanol

CH₃CH₂OH, EtOH ethanol

DMSO dimethyl sulfoxide

THF tetrahydrofuran

TEA Triethylamine

DMF N, N-dimethylformamide

NMP N-methylpyrrolidone

DMF-DMA N, N-dimethylformamide dimethyl acetal MTBE methyl tert-butyl ether

DPPA Azidophosphoric acid Diphenyl ester

CBZ, Cbz benzyloxycarbonyl

TFAA trifluoroacetic anhydride

DPEphos bis (2-diphenylphosphinophenyl) ether

DIPEA diisopropylethylamine, N, N-diisopropylethylamine

t-BuOH tert-butanol

BnOH benzyl alcohol

BH₃-THF solution borane in tetrahydrofuran

K₂CO₃Potassium carbonate

Na₂SO₄Sodium sulfate

NaN₃Sodium azide

KNO₃Potassium nitrate

NaOH sodium hydroxide

TsCl benzenesulfonyl chloride

H₂O water

Et₂O Ether

CH₃CN acetonitrile

NBS N-bromosuccinimide

Yb(OTf)₃Ytterbium trifluoromethanesulfonate

TFAA trifluoroacetic anhydride

MsCl methanesulfonyl chloride

Pd/C Palladium/carbon

N₂Nitrogen gas

L liter

mL of

uM, μ M micromole/L

uL, uL. mu.l

g

mg of

mmol millimole

mol mole of

M mol/l

DEG C

h hours

min, min

equiv. equivalent

TLC thin layer chromatography

Volume ratio of V/V, V/V

The following synthetic schemes describe the steps for preparing the compounds disclosed herein. Unless otherwise indicated, R^1C、R²、R^2a、R⁴、M¹、M²、M³、M⁴、M⁵L has the definition as described in the present invention.

The invention also provides a preparation method of the benzimidazole compound shown in the formula (I), and the following synthetic scheme describes the steps of preparing the compound disclosed by the invention. Unless otherwise indicated, R^N、R^CHaving the definitions as described in the present invention.

The synthesis scheme is as follows:

the compounds of formula (I) may be prepared by general synthetic methods as described in the synthetic schemes, with reference to the examples for the specific procedures, wherein Hal is halogen, preferably chlorine, bromine. The compound shown as the formula (I-A) and the compound shown as the formula (I-B) or the salt of the compound shown as the formula (I-B) are catalyzed (a catalytic system is Pd₂(dba)₃/XantPhos/CsCO₃Etc.) in a suitable solvent (e.g., 1, 4-dioxane, n-butanol, etc.) to obtain the compound of formula (I).

When R is^CIs hydrogen, R^NSelected from hydrogen, methyl or isopropyl, the synthesis of the compound (I-B) comprises the following steps:

1) the reaction of o-diphenylamine and nitrile bromide in a mixed solvent of methanol and water to obtain a cyclic product (a).

2) Cyclization product (a) with iodo compound R^NAnd I, reacting in the presence of potassium hydroxide by using acetone as a solvent to obtain a substituted compound (I-B-N) on the N position.

3) And carrying out coupling reaction on the compound (I-B-N) and the compound (I-A) to obtain a target compound.

When R is^NIs methyl, R^CWhen the substituent is the 6-position substituent, the method comprises the following steps:

1) reacting the compound (b-1) with a methylamine aqueous solution to obtain a compound (b-2);

2) hydrogenating and reducing the compound (b-2) under the catalysis of palladium carbon to obtain a compound (b-3);

3) the compound (B-3) reacts with nitrile bromide in a mixed solvent of methanol and water to obtain a cyclization product (I-B-C)₆)

When R is^NIs methyl, R^CWhen the substituent is a 7-position substituent, the method comprises the following steps:

1) reacting the compound (c-1) with methyl iodide in tetrahydrofuran under the action of sodium hydrogen to obtain a compound (c-2);

2) hydrogenating and reducing the compound (c-2) under the catalysis of palladium carbon to obtain a compound (c-3);

3) the compound (C-3) reacts with nitrile bromide in a mixed solvent of methanol and water to obtain a cyclization product (I-B-C)₇)

The benzimidazole compounds of the present invention are described in further detail with reference to specific examples.

Examples

Example 1: 2- ((2- ((1H-benzo [ d ] imidazol-2-yl) amino) -5-chloropyridin-4-yl) amino) -N-methoxybenzamide

Step 1: 1H-benzo [ d ] imidazol-2-amine

To a 50mL single neck pear flask were added 2.0g (18mmol) o-phenylenediamine and 10mL methanol solution (VMeOH/VH2O ═ 1:1), stirred at room temperature, 3.0g (28mmol) nitrile bromide was added slowly, the condenser tube was connected (deflated balloon), the flask was put into an oil bath at 60 ℃ and stirred overnight, and the color of the mixture gradually darkened to brown. The oil bath was removed and cooled to room temperature, TLC checked for complete reaction, methanol was removed, pH adjusted to 8 with 1M NaOH solution, extracted with EA (15mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and flash silica gel column chromatography (gradient elution polarity: PE: EA: 50:1 to EA: EtOH: 1) afforded about 2.4g of a pale yellow solid with 97% yield, Rf ═ 0.3(DCM/MeOH ═ 10/1). LC-MS [ M + H ] ═ 134.2.

Step 2: 2- ((2- ((1H-benzo [ d ] imidazol-2-yl) amino) -5-chloropyridin-4-yl) amino) -N-methoxybenzamide

The compound 2- [ (2, 5-dichloropyridin-4-yl) amino ] -N-methoxybenzamide (200mg, 0.64mmol), 1H-benzo [ d ] imidazol-2-amine (110mg,0.83mmol), cesium carbonate (417mg, 1.28mmol), XantPhos (93mg,0.16mol), Pd2(dba)3(117mg,0.128mmol) and 10ml of 1, 4-dioxane were added to a 20ml microwave tube, and under the protection of nitrogen, air in the system was pumped out by an oil pump, replaced with nitrogen, and the process was repeated three times, the microwave tube was sealed, and the reaction was carried out in a microwave reactor at 150 ℃ for 50 min. After the TLC detection reaction was complete, the catalyst was filtered off with celite, eluted with ethyl acetate, the solvent was dried by spin-drying, and flash column chromatography was performed (gradient elution polarity: DCM: MeOH: 100:1 to 40:1) to collect 67mg of a brown-yellow solid in 25.6% yield, Rf: 0.3 (DCM/MeOH: 5/1). 1H NMR (600MHz, DMSO) δ 11.97(s,1H),11.76(s,1H),10.36(s,1H),9.73(s,1H),8.18(s,1H),7.70(d, J ═ 7.2Hz,1H),7.62(dd, J ═ 9.2,7.2Hz,1H),7.45(brs,1H),7.31(brs,1H),7.20(t, J ═ 7.5Hz,1H),7.02(brs,1H),3.72(s,3H). HPLC purity: 96.34 percent. LC-MS [ M + H ] ═ 409.1.

Example 2: 2- ((5-chloro-2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

Step 1: 1H-benzo [ d ] imidazol-2-amine

Step 2: 1-methyl-1H-benzo [ d ] imidazol-2-amine

In a 50mL single-neck pear-shaped bottle, 0.55g (4.1mmol) of 1H-benzo [ d ] imidazol-2-amine is dissolved in 15mL of acetone, 0.46g (8.2mmol) of potassium hydroxide powder is added, stirring is carried out at room temperature for 10min, the mixture is moved to an ice-water bath, 0.28mL of methyl iodide (4.5mmol) is added dropwise, stirring is carried out for 10min, and the reaction is detected to be complete by TLC. After the mixture was cooled to room temperature, 15mLH2O was added, the solvent was removed, EA (10mL × 3) was used for extraction, the organic phases were combined, concentrated, and flash column chromatography was performed on silica gel (eluent: PE: EA ═ PE. 50:1) to obtain 550mg of a fluorescent brown viscous liquid with a yield of 90%, Rf ═ 0.25(DCM/MeOH ═ 5/1). LC-MS [ M + H ] ═ 148.1.

And step 3: 2- ((5-chloro-2- ((1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

200mg (0.64mmol) of the compound 2- [ (2, 5-dichloropyridin-4-yl) amino ] -N-methoxybenzamide, 141mg (0.95mmol) of 1-methyl-1H-benzo [ d ] imidazol-2-amine, 417mg (1.28mmol) of cesium carbonate, 93mg (0.16mol) of Xantphos, 117mg (0.128mmol) of Pd2(dba)3 and 10ml of 1, 4-dioxane were placed in a 20ml microwave tube, and under the protection of nitrogen, the air in the system was pumped off by means of an oil pump, replaced with nitrogen, and the process was repeated three times, the microwave tube was sealed and the reaction was carried out in a 150 ℃ microwave reactor for 50 min. After TLC detection reaction is completed, the catalyst is filtered out by using diatomite, the ethyl acetate is used for elution, the solvent is dried in a spinning mode, and the crude product is collected by fast silica gel column chromatography (gradient elution polarity: PE: EA is 100: 1-EA). The sample was dissolved in 2mL of dichloromethane, and an off-white solid was precipitated, filtered, and washed with dichloromethane to obtain 95.4mg of a pure product in 35.2% yield, Rf ═ 0.3(EA), and LC-MS [ M + H ] ═ 425.2. 1H-NMR (400MHz, d4-MeOH) δ 8.13(s,1H),7.76(d, J ═ 8.2Hz,1H),7.60(dd, J ═ 16.0,8.0Hz,2H),7.44-7.34(m,2H),7.31(d, J ═ 6.8Hz,1H),7.19(p, J ═ 7.2Hz,3H),3.82(s,3H),3.67(s, 3H). HPLC purity: 92.10 percent.

Example 3: 2- ((5-chloro-2- ((6-methoxy-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

Step 1: 5-methoxy-N-methyl-2-nitroaniline

In a 50mL single-necked pear-shaped flask, 40% methylamine water solution (6.0g,75.96mmol) was slowly added dropwise to a solution of 2-fluoro-4-methoxynitrobenzene (1g,5.84mmol), K2CO3(1.62g,11.69mmol) in 6mL DCM under ice-cooling, and stirred at room temperature overnight. After completion of the TLC reaction, 10mL of water was added, extracted with DCM (10mL x 3), the organic phases were combined and concentrated to give 1.1g of a bright yellow solid which was directly taken to the next reaction. Rf is 0.27(PE/EA 10/1) and LC-MS [ M + H ] is 183.2.

Step 2: 5-methoxy-N1-methylbenzene-1, 2-diamine

In a 50mL single-neck pear-shaped bottle, 5-methoxy-N-methyl-2-nitroaniline (600mg,3.29mmol) and 10% Pd/C (105mg,0.099mmol) were dissolved in 15mL ethanol, the oil-well pump was pumped out of the system under hydrogen protection, the air in the system was replaced three times with hydrogen, and the mixture was stirred overnight with a hydrogenation balloon at room temperature. TLC detection reaction is complete, the catalyst is filtered by diatomite, the ethyl acetate is washed, the solvent is dried by spinning, brown black viscous liquid is obtained, and the next step of reaction is directly carried out without post-treatment. Rf is 0.14(PE/EA 10/1). LC-MS [ M + H ] ═ 153.3.

And step 3: 6-methoxy-1-methyl-1H-benzo [ d ] imidazol-2-amine

In a 50mL single-neck eggplant-type bottle, 5-methoxy-N1-methylbenzene-1, 2-diamine (500mg,3.28mmol) and cyanogen bromide (521mg,4.93mmol) were added to 10mL of an aqueous methanol solution (v/v ═ 1:1), respectively, and the mixture was stirred at 60 ℃ for 4 hours. The reaction was run to completion by TLC, methanol was removed, adjusted to pH 8 with 1M NaOH solution, and extracted with EA (10mL × 3). The combined organic phases were concentrated by rotary evaporation and flash column chromatography on silica gel (gradient elution polarity: DCM: MeOH: 100:1 to 10:1) afforded approximately 484mg of a brown solid in 83.2% yield. Rf 0.21(DCM/MeOH 10/1). LC-MS [ M + H ] ═ 178.3. 1H-NMR (400MHz, CDCl3) δ 7.29(d, J ═ 8.6Hz,1H),6.74(dd, J ═ 8.6,2.2Hz,1H),6.63(d, J ═ 2.2Hz,1H),3.84(s,3H),3.50(s, 3H).

And 4, step 4: 2- ((5-chloro-2- ((6-methoxy-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

The compounds 2- [ (2, 5-dichloropyridin-4-yl) amino ] -N-methoxybenzamide (274mg,0.88mmol), 6-methoxy-1-methyl-1H-benzo [ d ] imidazol-2-amine (120mg,0.68mmol), cesium carbonate (440mg,1.35mmol), Xantphos (100mg,0.17mol), Pd2(dba)3(125mg,0.14mmol) and 10ml of 1, 4-dioxane were added to a 20ml microwave tube, and under nitrogen protection, the air in the system was pumped out by an oil pump, replaced with nitrogen, and the reaction was repeated three times, the microwave tube was sealed and the reaction was carried out in a 150 ℃ microwave reactor for 50 min. After TLC detection reaction is completed, the catalyst is filtered out by using kieselguhr, the ethyl acetate is used for elution, the solvent is dried in a spinning mode, and the crude product is subjected to silica gel column chromatography (gradient elution polarity: DCM: MeOH: 100: 0-50: 1) to obtain yellow powder of the crude product. The sample was dissolved in 5mL of dichloromethane, crystallized at low temperature, filtered with suction and washed with a small amount of DCM to give 72.5mg of yellow powder in 23.6% yield, Rf 0.25(DCM/MeOH 10/1). 1H-NMR (400MHz, DMSO) δ 12.01(s,1H),9.75(s,1H),9.64(s,1H),8.34(s,1H),8.14(s,1H),7.78(s,1H),7.62(d, J ═ 6.9Hz,2H),7.24(d, J ═ 8.5Hz,1H),7.16(t, J ═ 7.3Hz,1H),6.97(s,1H),6.70(s,1H),4.10(q, J ═ 5.2Hz,1H),3.79(s,3H),3.71(s,3H),3.66(s,2H),3.17(d, J ═ 5.2Hz,3H).

HPLC purity 98.73%. LC-MS [ M + H ] ═ 453.1.

Example 4: 2- ((5-chloro-2- ((6-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

Step 1: 5-fluoro-N-methyl-2-nitroaniline

In a 50mL single-necked pear-shaped flask, 40% aqueous methylamine solution (3.0g,39mmol) was slowly added dropwise to 2, 4-difluoronitrobenzene (2g,12.6mmol) under ice-bath to give a large amount of yellow solid, which was stirred for 1.5 h. And (3) detecting the reaction by TLC (thin layer chromatography), adding 40mL of water, precipitating a yellow solid, performing suction filtration, washing the solid with 10mL of water, collecting the solid, and spin-drying the solvent to obtain a crude product which is directly put into the next step. Rf is 0.45(PE/EA 10/1).

Step 2: 5-fluoro-N1-methylbenzene-1, 2-diamine

In a 50mL single-neck pear-shaped bottle, 5-fluoro-N-methyl-2-nitroaniline (2.1g,12mmol) and 10% Pd/C (390mg,0.37mmol) were dissolved in ethanol (50mL), the oil-well pump was pumped out under hydrogen protection, the air in the system was replaced three times with hydrogen, and the mixture was stirred overnight with a hydrogenation balloon at room temperature. TLC detection reaction is complete, the catalyst is filtered by diatomite, the ethyl acetate is washed, the solvent is dried by spinning, and brown-black viscous liquid 1.82g is obtained and is directly put into the next step of reaction without post-treatment. Rf is 0.12(PE/EA 10/1).

And step 3: 6-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-amine

In a 50mL single-neck eggplant-type bottle, 5-fluoro-N1-methylbenzene-1, 2-diamine (500mg,3.57mmol) and cyanogen bromide (566mg,5.35mmol) were added to 10mL of an aqueous methanol solution (v/v ═ 1:1), respectively, and the mixture was stirred at 60 ℃ overnight. And (5) detecting by TLC, and completing the reaction. Methanol was removed by spinning, the pH was adjusted to 8 with 1M NaOH solution, and extracted with EA (10mL × 3). The combined organic phases were concentrated by rotary evaporation and flash column chromatography on silica gel (gradient elution polarity: DCM: MeOH: 100:1 to 10:1) afforded about 446.4mg of a brown solid in 75.8% yield. Rf 0.4(DCM/MeOH 10/1). LC-MS [ M + H ] ═ 166.1. 1H-NMR (400MHz, CDCl3) δ 7.33(dd, J ═ 8.6,4.7Hz,1H),6.86(m,2H),4.64(s,2H),3.55(s, 3H).

And 4, step 4: 2- ((5-chloro-2- ((6-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

The compounds 2- [ (2, 5-dichloropyridin-4-yl) amino ] -N-methoxybenzamide (300mg,0.84mmol), 6-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-amine (120mg,0.73mmol), cesium carbonate (470mg,1.45mmol), Xantphos (105mg,0.18mol), Pd2(dba)3(133mg,0.15mmol) and 10ml of 1, 4-dioxane were added to a 20ml microwave tube, and under nitrogen protection, the air in the system was pumped out by an oil pump, replaced with nitrogen, and the reaction was repeated three times, the microwave tube was sealed and the reaction was carried out in a 150 ℃ microwave reactor for 50 min. After TLC detection reaction is completed, the catalyst is filtered out by using kieselguhr, the ethyl acetate is used for elution, the solvent is dried in a spinning mode, and the crude product is subjected to silica gel column chromatography (gradient elution polarity: DCM: MeOH: 100: 0-50: 1) to obtain yellow powder of the crude product. The sample was dissolved in 5mL of dichloromethane to precipitate an off-white solid, which was filtered and washed with 10mL of dichloromethane to give 173.3mg of pure product in 54.1% yield, Rf 0.45(DCM/MeOH 10/1). 1H-NMR (400MHz, DMSO) δ 12.04(s,1H),9.79(s,1H),8.20(s,1H),7.80(s,1H),7.66(d, J ═ 7.6Hz,2H),7.41-7.26(m,2H),7.21(t, J ═ 7.4Hz,1H),6.95(t, J ═ 8.4Hz,1H),3.75(s,3H),3.68(s,3H), HPLC purity 96.22%. LC-MS [ M + H ] ═ 441.2.

Example 5: 2- ((5-chloro-2- ((7-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

Step 1: 2-fluoro-N-methyl-6-nitroaniline

NaH (140mg,3.52mmol) was added to a solution of 2-fluoro-6-nitroaniline (500mg,3.20mmol) in 10mL THF in a 50mL single-necked pear-shaped flask under ice-bath, stirred for 10min, a solution of iodomethane (0.2mL,3.20mmol) in 1.0mL THF was added dropwise slowly and stirred for 30 min. The mixture was cooled to room temperature and stirred at room temperature. The reaction was completed by TLC and 10mLH2O was added to quench the reaction, extracted with EA (8mL × 3), the organic phases combined, concentrated and chromatographed on silica gel (elution polarity: PE: EA ═ 100:1) to give 512.8mg of red brown liquid in 94.1% yield and Rf ═ 0.9(PE/EA ═ 10/1). 1H-NMR (400MHz, CDCl3) δ 7.96(d, J ═ 8.7Hz,1H),7.85(s,1H),7.20(dd, J ═ 13.8,7.8Hz,1H),6.57(td, J ═ 8.3,4.6Hz,1H),3.27(dd, J ═ 7.4,5.6Hz,3H), LC-MS [ M + H ] ═ 171.1.

Step 2: 6-fluoro-N1-methylbenzene-1, 2-diamine

Dissolving 2-fluoro-N-methyl-6-nitroaniline (500mg,2.94mmol) and 10% Pd/C (93mg,0.088mmol) in ethanol (10mL) in a 50mL single-neck pear-shaped bottle, under the protection of hydrogen, pumping an oil pump, discharging air in the system, replacing the air with hydrogen for three times, and adding hydrogen to stir for 2 hours by a balloon at room temperature. TLC detection reaction is complete, the catalyst is filtered by diatomite, the ethyl acetate is washed, the solvent is dried by spinning, 372mg of brown liquid is obtained, the yield is 90.4%, and the next step of reaction is directly carried out without post-treatment. Rf is 0.2(PE/EA 10/1). LC-MS [ M + H ] ═ 141.25.

And step 3: 7-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-amine

In a 50mL single-neck eggplant-type bottle, 6-fluoro-N1-methylbenzene-1, 2-diamine (370mg,2.64mmol) and cyanogen bromide (420mg,3.96mmol) were added to 10mL of an aqueous methanol solution (v/v ═ 1:1), respectively, and the mixture was stirred at 60 ℃ overnight. And (5) detecting by TLC, and completing the reaction. Methanol was removed by spinning, the pH was adjusted to 8 with 1M NaOH solution, and extracted with EA (10mL × 3). The combined organic phases were concentrated by rotary evaporation and flash column chromatography on silica gel (gradient elution polarity: DCM: MeOH: 100:1 to 20:1) afforded 368mg of white solid in 84.4% yield. Rf 0.3(DCM/MeOH 10/1). LC-MS [ M + H ] ═ 166.1. 1H-NMR (400MHz, CDCl3) δ 7.17(d, J ═ 7.9Hz,1H),7.00(td, J ═ 8.0,5.2Hz,1H),6.76(dd, J ═ 11.6,8.2Hz,1H),4.99(s,2H),3.74(s,3H).

And 4, step 4: 2- ((5-chloro-2- ((7-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

The compounds 2- [ (2, 5-dichloropyridin-4-yl) amino ] -N-methoxybenzamide (294mg,0.94mmol), 7-fluoro-1-methyl-1H-benzo [ d ] imidazol-2-amine (120mg,0.73mmol), cesium carbonate (470mg,1.45mmol), Xantphos (105mg,0.18mol), Pd2(dba)3(133mg,0.15mmol) and 10ml of 1, 4-dioxane were added to a 20ml microwave tube, and under nitrogen protection, the air in the system was pumped out by an oil pump, replaced with nitrogen, and the reaction was repeated three times, the microwave tube was sealed and the reaction was carried out in a 150 ℃ microwave reactor for 50 min. After TLC detection reaction is completed, the catalyst is filtered out by using kieselguhr, the ethyl acetate is used for elution, the solvent is dried in a spinning mode, and the crude product is subjected to silica gel column chromatography (gradient elution polarity: DCM: MeOH: 100: 0-50: 1) to obtain yellow powder of the crude product. The sample was dissolved in 5mL of dichloromethane to precipitate a yellow solid, which was filtered and washed with 10mL of dichloromethane to afford 142mg of pure product in 44.3% yield, Rf 0.3(DCM/MeOH 20/1). 1H-NMR (400MHz, DMSO) δ 12.00(s,1H),9.83(d, J ═ 19.9Hz,2H),8.39(s,1H),8.19(s,1H),7.80(s,1H),7.63(d, J ═ 7.8Hz,2H),7.18(dd, J ═ 11.6,7.7Hz,2H),7.11-6.97(m,1H),6.98-6.84(m,1H),3.84(s,3H),3.71(s, 3H). HPLC purity 95.66%. LC-MS [ M + H ] ═ 441.2.

Example 6: 2- ((5-chloro-2- ((1-isopropyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

Step 1: 1-isopropyl-1H-benzo [ d ] imidazol-2-amine

In a 50mL single-necked eggplant-type bottle, 0.55g (4.1mmol) of 1H-benzo [ d ] imidazol-2-amine

Dissolved in 15mL of acetone, added with 0.70g (12mmol) of potassium hydroxide powder and 1.1g (8.2mmol) of potassium carbonate, stirred at room temperature for 10min, transferred to an ice-water bath, added dropwise with 0.45mL (4.5mmol) of 2-iodopropane, transferred to a 65 ℃ oil bath and stirred for 3 h. The reaction was checked by TLC, the reaction was diluted with EA, the solid was filtered off with suction, the reaction was freed of solvent, 10mL of water was added, extracted with EA (8mL × 3, the organic phases were combined, dried over Na2SO4, concentrated, and purified by flash silica gel column chromatography (gradient elution DCM: MeOH ═ 100:1 to 30:1) to give 275mg of a pale yellow solid with a yield of 38%, Rf ═ 0.25(DCM/MeOH ═ 5/1), 1H-NMR (400MHz, CDCl3) δ 7.40(d, J ═ 7.8Hz,1H),7.27(d, J ═ 7.4Hz,1H),7.11(t, J ═ 7.3Hz,1H),7.04(t, J ═ 7.6Hz,1H),4.45(dt, J ═ 13.9,6.9Hz,1H),1.59(d, J ═ 6.9, 7.9H), 1.29H ═ 1H, 29.176H, MS ═ 1H + 1H.

Step 2: 2- ((5-chloro-2- ((1-isopropyl-1H-benzo [ d ] imidazol-2-yl) amino) pyridin-4-yl) amino) -N-methoxybenzamide

200mg (0.64mmol) of the compound 2- [ (2, 5-dichloropyridin-4-yl) amino ] -N-methoxybenzamide, 168mg (0.96mmol) of 1-isopropyl-1H-benzo [ d ] imidazol-2-amine, 417mg (1.28mmol) of cesium carbonate, 93mg (0.16mol) of Xantphos, 117mg (0.128mmol) of Pd2(dba)3 and 10ml of 1, 4-dioxane were placed in a 20ml microwave tube, and under the protection of nitrogen, the air in the system was pumped off by means of an oil pump, replaced with nitrogen, and the reaction was repeated three times, the microwave tube was sealed and the reaction was carried out in a 150 ℃ microwave reactor for 50 min. After TLC detection reaction is completed, the catalyst is filtered out by using diatomite, ethyl acetate elution is carried out, the solvent is dried by spinning, and the crude product of 109mg is collected by fast silica gel column chromatography (gradient elution polarity: PE: EA is 100: 1-EA), the yield is 37.7%, and Rf is 0.4 (EA). Recrystallization from DCM/PE gave 22mg of a pale yellow powder, LC-MS [ M + H ] ═ 451.1. 1H-NMR (400MHz, CDCl3) δ 9.09(s,1H),8.08(s,1H),7.66(d, J ═ 7.2Hz,1H),7.45-7.50(m,2H),7.26(s,2H),7.00-7.10(m,4H),5.12(brs,1H),3.80(s,3H),1.56(d, J ═ 5.6Hz, 6H). HPLC purity: 96.15 percent.

Biochemical testing of FAK activity:

EXAMPLE 1 in vitro enzymatic inhibitory Activity of Compounds of the invention

FAK used in the experiments was purchased from Carna (Cat.No. 08-137) and Fluorescein-Poly GT from Invitrogen (Cat.No. PV3610). Experiment the Lantha screen method was used to determine the inhibitory activity of compounds against FAK.

The experimental method comprises the following steps:

1.1 Xkinase buffer preparation

1 Xpreparation of kinase buffer (25mM HEPES, pH7.5,0.01mM Triton,10mM MgCl)₂,0.5mMEGTA,0.01％BRIJ-35,2mM DTT)。

2. Compound preparation for kinase assay: serial dilution of compounds

(1) Diluting the test compound to 100 times of the highest test concentration by using 100% DMSO (the highest test concentration is 10uM, and the concentration of the compound prepared in the step is 1000 uM); (2) test compounds were transferred to one of the 96 wells and diluted in 4-fold gradients by adding 20uL of stock solution to 60uL of 100% DMSO for a total of 10 concentrations; (3) 100uL of 100% DMSO was added to each of the empty wells of the same 96-well plate, and the plate was labeled as the source plate as the no-compound and no-enzyme control for the experiment; (4) preparing a 96-well intermediate plate, respectively taking 4uL of each concentration compound from a source plate, transferring the compound to the intermediate plate, adding 96uL of kinase buffer solution, and shaking and uniformly mixing for 10 minutes; (5) preparing an experimental plate: transfer 5uL from each concentration well of the intermediate plate to 384 well assay plates, two subpores per concentration.

3. Kinase reaction

(1) Preparation of 2 × kinase solution: preparing FAK solution (the final concentration of FAK is 0.4nM) with 2 times of the final test concentration by adopting 1 Xkinase buffer solution, transferring 5uL of kinase solution to corresponding holes of an experimental plate, adding 5uL of kinase buffer solution into a non-enzyme control hole, and vibrating the plate; (2) preparation of 4 × substrate solution: 1 Xkinase buffer solution is adopted to prepare 4 times of final test concentration fluoroescein-polyGT and ATP solution (the final concentration of fluoroescein-polyGT is 0.2uM, ATP 6uM), 2.5uL of substrate solution is taken to be transferred to corresponding holes of an experimental plate, kinase reaction is started, and the plate is shaken; (3) kinase reaction: incubate for 30min at room temperature in the dark.

4. Kinase detection

Preparing a detection solution with 2 times of final test concentration by adopting an antibody dilution buffer solution (the final concentration of the antibody is 2nM, and the final concentration of EDTA is 10mM), transferring 10uL of the detection solution to corresponding holes of an experimental plate, stopping kinase reaction, centrifugally mixing, incubating for 60 minutes, and detecting a fluorescent signal by a plate reading machine.

5. Data measurement

Fluorescence data were collected at 340nM excitation light and 520nM and 495nM emission light on Envision.

6. Fitting of curves

(1) Copying RFU data from the Envision program; (2) calculating the ratio of RFU 520nM/RFU 495 nM; (3) the ratio was converted to percent inhibition: inhibition = (max-sample value)/(max-min) × 100, where the maximum is DMSO control and the minimum is no enzyme control; (4) IC50 calculates the equation as:

Y＝Bottom+(Top-Bottom)/(1+(IC₅₀/X)^HillSlope)

specific IC₅₀The activity data are shown in Table 1.

TABLE 1 in vitro enzymatic inhibitory Activity of representative Compounds of the invention

Examples	IC₅₀(nM)	Examples	IC₅₀(nM)
				Example 1	18	Example 2	2.5
Example 3	1.7	Example 4	1.4
				Example 5	2.6	Example 6	2.2

As can be seen from table 1, (1) from the in vitro inhibitory activity data of examples 1,2, and 6, it can be preliminarily obtained that a substituent is introduced on N (i.e. 1 site) of the imidazole ring, which can significantly increase the in vitro inhibitory activity of FAK enzymology, and a group with larger steric hindrance is introduced to have stronger inhibitory activity;

(2) from the in vitro inhibitory activity data of examples 2,4 and 5, it can be obtained that the substituent introduced at the 6-position of the imidazole ring has stronger inhibitory activity;

(3) from the activity data of examples 2, 3 and 4, it can be seen that the substitution of the electron-deficient group at position 6 has a stronger activity.

In conclusion, the compound of the invention has large steric hindrance of the substituent at the 1 position and electron deficiency of the substituent at the 6 position, and is beneficial to increasing the inhibitory activity of the compound on FAK enzymology; meanwhile, the direction is provided for the subsequent structure and effect research.

EXAMPLE 2 inhibitory Activity of Compounds of the invention on cells in vitro under 2D, 3D conditions

The experimental method comprises the following steps:

cell experiment conditions:

cell name	Cell/well	Incubation time (h)	Complete culture medium
				BXPC-3	3000	96	RPMI1640+10％FBS
NCI-H1975	4000	96	RPMI1640+10％FBS

1) Spreading glue

a. Preparing complete culture medium, and mixing completely. b. Mixing matrigel with corresponding cell culture medium at a ratio of 1:1, uniformly mixing, uniformly paving the mixture at the bottom of a 96-well plate with a transparent bottom at 40 uL/hole, and then placing the plate in an incubator at 37 ℃ for solidification for later use.

2) Cell plating

a. Recovering the cells, and selecting cell strains with good growth state after two generations. b. Will be thinThe cell culture dish is taken out from the incubator, and the cell name, the type of the culture medium and the cell generation number marked on the flask are checked. c. The medium was discarded, rinsed with PBS, and cells were digested with pancreatin. d. Complete medium was added and transferred to a centrifuge tube and centrifuged at 1000rpm for 5 minutes. e. The cell supernatant in the centrifuge tube was discarded and the appropriate complete medium was added to resuspend the cells (2D: resuspend with complete medium; 3D: resuspend with complete medium containing 2% matrigel). f. Counting using a cell counter. g. The cell suspension is adjusted to the appropriate concentration. h. Adding the cell suspension into a 96-well plate (2D: 96-well plate with transparent bottom; 3D: 96-well plate obtained in step 1), labeling detailed information such as cell name, plate density and date at 90 uL/well, and placing the culture plate in CO₂The incubator was overnight.

3) Preparation and addition of Compounds

a. Preparation of compounds (test compounds dissolved in DMSO, 10mM stock solution): the compound is diluted 3 times by DMSO to obtain 9 concentration gradients, the gradient diluted compound is diluted 20 times by complete culture medium, and the mixture is mixed uniformly to obtain 10 × concentration drug working solution. b. Addition of the compound: the cell culture plate was removed, 10 uL/well of the above 10 Xconcentration of the drug working solution was added to the corresponding well of the cell culture plate, and incubated at 37 ℃ for 96 hours in an incubator.

4) Detection and analysis

a. After the compound is treated for 96 hours, the cell morphology is observed under an inverted microscope, the cell growth state in the DMSO control hole is normal, and no pollution phenomenon is seen. b. The CCK-8 reagent was allowed to equilibrate for 30 minutes at room temperature. c. After the cell incubation was completed, 10 uL/well of CCK-8 reagent was added thereto. d. The cell culture plate was placed in an incubator at 37 ℃ and incubation continued for 2-4 hours. e. The Optical Density (OD) at a wavelength of 450nm was read for each well using a microplate reader. f. The results of the analysis are recorded: the data were analyzed using GraphPadprism 5.0 software, and the formula for the inhibition of cell growth was as follows, IC₅₀It can be calculated automatically in GraphPadPrism 5.0.

Growth inhibition ratio%_{Negative group}-OD_{Experimental group})/(OD_{Negative group}-OD_{Blank group})*100％

TABLE 2 in vitro cytological inhibitory Activity of representative Compounds of the invention

As can be seen from Table 2, the compound of the present invention has stronger cell growth inhibition effect under 3D condition, and the cell growth inhibition effect of the compound of the present invention is obviously better than that of GSK 2256098.

EXAMPLE 3 pharmacokinetic Activity of Compounds of the invention

The experimental method comprises the following steps:

1. preparation of test Compound solution

The compound to be tested is prepared into solution by DMSO, KolliphorHS15 and Saline according to a certain proportion, and is used for oral administration and intravenous injection administration.

2. Animal experiments

Taking 190g male SD rats 140-; tail vein blood was collected at time points 0.083, 0.25, 0.5, 1,2, 4, 6,8, and 24 hours after intravenous administration; tail vein blood was collected at time points 0.25, 0.5, 1,2, 4, 6,8 and 24 hours after oral administration. A standard curve of the appropriate range was established based on the sample concentration, and the concentration of the test compound in the plasma sample was determined in MRM mode using LC-MS/MS model AB SCIEX API 4000. Pharmacokinetic parameters were calculated according to the drug concentration-time curve using the winnonlin6.3 software non-compartmental model method.

3. Results

TABLE 3 pharmacokinetic Activity of representative Compounds of the invention

Remarking: "/" indicates not calculated.

As shown in Table 3, the compounds of the present invention have good in vivo metabolism, good absorption, high exposure and high bioavailability.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A benzimidazole compound having a structure represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite, prodrug, or mixture thereof:

wherein,

R^Nselected from hydrogen, methyl, isopropyl;

R^Cis selected fromHydrogen, methoxy, fluoro.

2. The benzimidazole compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite, prodrug or mixture thereof according to claim 1, wherein R is selected from the group consisting of^NSelected from methyl, isopropyl;

R^Cselected from hydrogen, methoxy, fluorine.

3. The benzimidazole compound of claim 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite, prodrug or mixture thereof, wherein the benzimidazole compound has the structure of formula (II):

wherein,

R^Nselected from methyl, isopropyl;

R^Cselected from hydrogen and fluorine.

4. The benzimidazole compound of claim 2, or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite, prodrug or mixture thereof, wherein the benzimidazole compound has the structure of formula (III):

wherein,

R^Nis selected from methyl;

R^Cselected from methoxy and fluorine.

5. The benzimidazole compound of claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite, prodrug or mixture thereof, wherein the benzimidazole compound has one of the following structures:

6. a medicament comprising a benzimidazole compound of any one of claims 1-5, or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite, prodrug or mixture thereof, and a pharmaceutically acceptable excipient.

7. The medicament of claim 6, wherein the active ingredient of the medicament further comprises an additional therapeutic agent.

8. The medicament of claim 7, wherein the additional therapeutic agent comprises one or more of an anticancer agent and a drug for treating pulmonary hypertension.

9. Use of a compound according to any one of claims 1 to 5 for the preparation of a medicament for the prophylaxis and treatment of FAK-related diseases.

10. The use according to claim 9, wherein the FAK-related disease comprises: cancer, pulmonary hypertension, or pathological angiogenesis.