WO2023187471A1 - Heteroaryl derivative compounds, and uses thereof - Google Patents

Abstract

The present invention relates to a heteroaryl derivative and uses thereof. The heteroaryl derivative compound of the present invention exhibits excellent inhibitory activity against RAF, and thus may be useful for the treatment or prevention of RAF-related diseases.

Description

HETEROARYL DERIVATIVE COMPOUNDS, AND USES THEREOF

RELATED APPLICATIONS

This application claims priority to KR 10-2022-0039695, filed March 30, 2022, and to KR10-2022-0109679, filed August 31, 2022. Each of these is incorporated herein by reference in its entirety.

FIELD

The present invention relates to a heteroaryl derivative compound and medicinal uses thereof. Specifically, the present invention relates to a heteroaryl derivative compound having RAF inhibitory activity.

BACKGROUND

The RAS/RAF/MEK/ERK protein kinase signaling pathway plays a very important role in the regulation of cellular function, and is specifically involved in cell proliferation, differentiation, survival, and angiogenesis (Biology of the Cell, 2001, 93, 53-62). In the signaling pathway, when guanosine triphosphate (GTP) is bound to the RAS protein, phosphorylation and activation of the RAF protein in the protoplasmic membrane proceeds. Subsequently, the activated RAF protein phosphorylates and activates the MEK protein, and the MEK protein phosphorylates and activates the ERK protein. Translocation of activated ERK from the cytoplasm to the nucleus results in regulation and phosphorylation of transcription factors such as Elk-1 and Myc.

RAF protooncogenes are serine/thr protein kinases that transmit signals from growth factor receptors activated in the cell membrane to transcription factors in the nucleus. The activation of the RAF protein is accompanied by phosphorylation of tyrosine, serine, and threonine residues of RAF protein, and direct phosphorylation by receptor tyrosine kinase or phosphorylation by protein phosphorylation enzymes controlled by these receptors is known as the mechanism of RAF activation. Among them, when controlled by a receptor, RAS is involved in the activation of RAF. Signals reaching RAF are then transferred to the nucleus via a signaling pathway leading to the RAF/MEK/ERK protein kinase. In this signaling pathway, a series of kinases are arranged into species to transmit signals, which play an essential role in cell growth and differentiation (Nature Rev. Mol. Cell. Biol., 2004, 5, 875-885), and the activity of RAF/MEK/ERK has been reported to be upregulated in a number of factor-dependent tumors.

As such, RAF acts as a major propagator of RAS function, providing a theoretical background for chemotherapy in the case of cancer with mutations or activations of RAS in inhibiting the action of this protein. RAF proteins have three isoforms of ARAF, BRAF, and CRAF (also known as RAF-1) with three functions(Biochim. Biophys. Acta., 2003, 1653, 25-40), all three RAF genes are expressed in most tissues, and high expression of BRAF occurs in neurocellular tissues and ARAF occurs in urinary reproductive tissues. Although each RAF family has a very similar amino acid sequence, biochemical activity and biological functionality are distinguished from each other (Exp. Cell. Res. 1999, 253, 34-46).

Studies have shown that BRAF is an important isoform protein related to cell proliferation and is an important target of oncogenic RAS. Abnormal mutations in the body have only been identified in BRAF cases, and are known to occur at a frequency of 30-60% in malignant skin melanoma (Nature, 2002, 417, 949-954), 30-50% in thyroid cancer, 5-20% in colon cancer, and 30% or less in ovarian cancer (Nature Rev. Mol. Cell Biology, 20045, 875 and 885). So far, more than 45 BRAF mutations have been known, but the most frequent mutation is that valine number 600 mutates with glutamic acid (V600E), which is observed in more than 90% of human cancers. This mutation is believed to increase the kinase activity of BRAF and transmit RAF/MEK/ERK signals to sub-signaling pathways that include structural activity of ERK as a result of RAS and growth factor receptor activation.

Accordingly, as a result of careful efforts to develop a new drug capable of inhibiting the activity of RAF, the present inventors confirmed that the compounds of the present invention have an anticancer effect by inhibiting the activity of RAF, thereby completing the present invention.

PRIOR ART LITERATURE

Non-patent literature

(Non-patent literature 0001) Biology of the Cell, 2001, 93, 53-62

(Non-patent literature 0002) Nature Rev. Mol. Cell. Biol., 2004, 5, 875-885

(Non-patent literature 0003) Biochim. Biophys. Acta., 2003, 1653, 25-40

(Non-patent literature 0004) Exp. Cell. Res. 1999, 253, 34-46

(Non-patent literature 0005) Nature, 2002, 417, 949-954

(Non-patent literature 0006) Nature Rev. Mol. Cell Biology, 2004, 5, 875-885

DISCLOSURE

Technical Problem

An object of the present invention is to provide a heteroaryl derivative having a novel structure, an stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for preparing the heteroaryl derivative compound.

Still another object of the present invention is to provide a pharmaceutical use of the heteroaryl derivative compound, and specifically, to a pharmaceutical composition for the treatment or prevention of RAF-related diseases comprising the heteroaryl derivative compound as an active ingredient, use of the compound for the treatment or prevention of RAF-related diseases, or a method for treating or preventing RAF-related diseases comprising administering the compound.

Technical Solution

In order to achieve the above-described objects, the present inventors made efforts to study, and as a result, found that the following heteroaryl derivative compounds represented by Chemical Formula 1 inhibited the proliferation of RAF-activated cells, and completed the present invention.

Heteroaryl derivative compound

The present invention provides a compound represented by following Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

Chemical Formula 1

in the Chemical Formula 1 ,

X is CH₂ or O;

R is -C_1-6alkyl, -C_1-6haloalkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl in which at least one H of the aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, - C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo; a ring Y is aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl in which at least one H of the aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, - C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo;

Z₁ is CR₁, NR₂, or N; Z₂ is NR₂, N, O, or S;

Z₃ is CR₃ or N;

R₁ and R₂ are each independently -H, -C_1-6alkyl, -C_1-6hyroxy alkyl, -C_1-6aminoalkyl, -C_{1- 6}haloalkyl, -C_1-6alkyl-N(C_1-6alkyl)(C_1-6alkyl), -C_1-6alkyl-O-C_1-6alkyl, cycloalkyl or heterocycloalkyl, wherein at least one H of the cycloalkyl or heterocycloalkyl may be substituted with -C_1-6alkyl, -C_1-6haloalkyl, or -halo;

R₃ is -H or -C_1-6alkyl.

According to an embodiment of the present invention, the compound represented by Chemical Formula 1 above, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof may be included in the following scope:

X is CH₂ or O;

R is -C_1-6alkyl, -C_1-6haloalkyl, aryl, or heteroaryl, wherein at least one H of the aryl or heteroaryl may be substituted with -C_1-6alkyl, -C_1-6haloalkyl, -CN, or -halo; a ring Y is aryl or heteroaryl, wherein at least one H of the aryl or heteroaryl may be substituted with -C_1-6alkyl or -halo;

Z₁ is CR, NR₂, or N;

Z₂ is NR₂, N, or S;

Z₃ is CR₃ or N;

R₁ and R₂ are each independently -H, -C_1-6alkyl, -C_1-6alkyl-N(C_1-6alkyl)(C_1-6alkyl), -C_{1- 6}alkyl-O-C_1-6alkyl, 3- to 7-membered cycloalkyl or 3- to 7-membered heterocycloalkyl in which at least one H of the 3- to 7-membered cycloalkyl or 3- to 7-membered heterocycloalkyl may be substituted with -C_1-6alkyl or -halo;

R₃ is -H.

According to an embodiment of the present invention, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof may be selected from the group consisting of the compounds of Examples 1 to 60 described in Table 2.

In the present invention, unless otherwise specified, the term "alkyl" may refer to a straight or branched chain acyclic, cyclic, or saturated hydrocarbon to which they are bonded. For example, "C_1-6alkyl" may indicate an alkyl containing 1 to 6 carbon atoms. As an example, acyclic alkyl may include, but is not limited to, methyl, ethyl, n-propyl, n-butyl, isopropyl, sec-butyl, isobutyl, tert-butyl, or the like. Cyclic alkyl may be used interchangeably with “cycloalkyl” as used herein, and as an example, may include, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, or the like. In certain embodiments, the cycloalkyl is monocyclic. In certain embodiments, the cycloalkyl is 3- 7-membered.

In the present invention, “alkoxy” may indicate -(O-alkyl) as an alkyl ether group, wherein alkyl is the same as defined above. For example, “Ci-6alkoxy” may mean alkoxy containing C_{1 6}alkyl, that is, -(O-C_1-6alkyl), and as an example, may include, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec -butoxy, tert-butoxy, or the like.

In the present invention, “halo” may be F, Cl, Br, or I.

As used herein, "haloalkyl" may mean a straight or branched chain alkyl (hydrocarbon) having one or more halo-substituted carbon atoms as defined herein. Examples of the haloalkyl may include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl or n-butyl independently substituted with one or more halogens, such as F, Cl, Br, or I.

As used herein, "hydroxy alkyl" may indicate a straight or branched chain alkyl (hydrocarbon) having a carbon atom substituted with -hydroxy (-OH). Examples of the haloalkyl may include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl or n-butyl independently substituted with one or more halogens, such as -OH.

As used herein, "aminoalkyl" may mean a straight or branched chain alkyl (hydrocarbon) having a carbon atom substituted with amino-(NR'R"). Here, R' and R" may be each independently selected from the group consisting of hydrogen and C_1-6alkyl, and the selected R' and R" may be each independently substituted or unsubstituted.

In the present invention, "heterocycloalkyl" may mean a ring containing 1 to 5 heteroatoms selected from N, O and S as atoms forming the ring, and may be saturated or partially unsaturated. Here, when unsaturated, it may be referred to as a heterocycloalkene. Unless otherwise stated, heterocycloalkyl may be a single ring or a multiple ring such as a spiro ring, a bridged ring or a fused ring. In addition, "3- to 12-membered heterocycloalkyl" may indicate a heterocyclo alkyl containing 3 to 12 atoms forming a ring. As an example, the heterocycloalkyl may include, but is not limited to, pyrrolidine, piperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidin-2,4(1H,3H)-dione, 1,4- dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydro thiophene, quinuclidine, tropane, 2-azaspiro[3.3]heptane, (1R,5S)-3-azabicyclo[3.2.1]octane, (1s,4s)-2- azabicyclo[2.2.2]octane, or (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane, and the like.

In the present invention, "arene" may mean an aromatic hydrocarbon ring. The arene may be a monocyclic arene or a polycyclic arene. The number of ring-forming carbons in the arene may be 5 or more and 30 or less, 5 or more and 20 or less, or 5 or more and 15 or less. Examples of the arene may include, but are not limited to, benzene, naphthalene, fluorene, anthracene, phenanthrene, bibenzene, terbenzene, quaterbenzene, quinquebenzene, sexibenzene, triphenylene, pyrene, benzofluoranthene, chrysene, and the like. In the present specification, the residue obtained by removing one hydrogen atom from "arene" is referred to as "aryl".

In the present invention, "heteroarene" may be a ring containing at least one of O, N, P, Si, and S as a heterogeneous element. The number of ring-forming carbons in the heteroarene may be 2 or more and 30 or less, or 2 or more and 20 or less. The heteroarene may be a monocyclic heteroarene or a polycyclic heteroarene. The polycyclic heteroarene may have, for example, a bicyclic or tricyclic structure. Examples of the heteroarene may include thiophene, purine, pyrrole, pyrazole, imidazole, thiazole, oxazole, isothiazole, oxadiazole, triazole, pyridine, bipyridyl, triazine, acridyl, pyridazine, pyrazine, quinoline, quinazoline, quinoxaline, phenoxazine, phthalazine, pyrimidine, pyridopyrimidine, pyridopyrazine, pyrazinopyrazine, isoquinoline, indole, carbazole, imidazopyridazine, imidazopyridine, imidazopyrimidine, pyrazolopyrimidine, imidazopyrazine or pyrazolopyridine, N-arylcarbazole, N-heteroarylcarbazole, N-alkylcarbazole, benzoxazole, benzoimidazole, benzothiazole, benzocarbazole, benzothiophene, dibenzothiophene, thienothiophene, benzofuran, phenanthroline, isoxazole, oxadiazole, thiadiazole, benzothiazole, tetrazole, phenothiazine, dibenzosilole, dibenzofuran, and the like, but are not limited thereto. In an embodiment of the present invention, heteroarene may also include bicyclic heterocyclo-arene containing heteroarene fused to an arene ring or a cycloalkyl ring fused to heterocycloalkyl rings. In the present specification, the residue obtained by removing one hydrogen atom from the "heteroarene" is referred to as "heteroaryl".

The above-mentioned homogeneous or heterogeneous substituents may be substituted one or more at the same or different positions, and may be sequentially substituted. The meaning of "sequentially" means that in the formula, one substituent is substituted and then another substituent is successively substituted in the substituent, for example, a cycloalkyl group is substituted in the alkyl group after the alkyl group is substituted, and the When a carbonyl group is sequentially substituted for a cycloalkyl group, it can be indicated that the cycloalkyl group is sequentially substituted by naming it carbonylcycloalkylalkyl.

In addition, the connection radicals listed above do not specify the coupling direction, and the coupling direction is arbitrary. For example, the radical L connected in

can be -M-W-, where ring A and ring B can be connected in the same direction as the reading order from left to right to form and ring A and ring B can be connected in the

opposite direction to the reading order from left to right to form

In the present invention, the term "stereoisomers (e.g., enantiomers)" mean compounds of the present invention or salts thereof that have the same chemical formula or molecular formula but are different in stereostructure. Each of these enantiomers and mixtures thereof are also included within the scope of the present invention. Unless otherwise specified, the straight solidline bond (-) connecting an asymmetric carbon atom may include a wedge-shaped solid-line bond or a wedge-shaped dashed-line bond

indicating the absolute configuration of the stereocenter. The term “stereoisomer” and “optical isomer” are used interchangeably.

The compound of Chemical Formula 1 of the present invention may exist in the form of a "pharmaceutically acceptable salt". As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The term "pharmaceutically acceptable salt" as used herein means any and all organic or inorganic acid addition salts of the compound represented by Chemical Formula 1 of which side effects caused by the salt do not reduce the beneficial efficacy of the compound at concentrations having an effective action that is relatively non-toxic and harmless to a patient.

Acid addition salts are prepared by conventional methods, for example by dissolving the compound in an excess of aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. An acid or alcohol in an equimolar amount of the compound and water may be heated, and the mixture may then be evaporated to dryness, or the precipitated salt may be filtered off with suction.

Here, an organic acid and an inorganic acid may be used as the free acid, wherein the inorganic acid may be hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, or the like, and the organic acid may be methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, or the like. However, the present invention is not limited thereto.

In addition, it is possible to prepare a pharmaceutically acceptable metal salt using a base. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. Here, it is pharmaceutically suitable to prepare a sodium, potassium, or calcium salt as the metal salt, but the present invention is not limited thereto. Further, the corresponding silver salt may be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Unless otherwise indicated, the pharmaceutically acceptable salt of the present invention includes salts of acidic or basic groups that may be present in the compound of Chemical Formula 1. For example, the pharmaceutically acceptable salt may include sodium, calcium and potassium salts of hydroxyl groups, and the like, and as other pharmaceutically acceptable salts of amino groups, may include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate(mesylate), and p-toluenesulfonate (tosylate) salts, and the like, and may be prepared by a method for preparing a salt known in the art.

In certain embodiments, X is O. In certain embodiments, X is CH₂.

In certain embodiments, R is phenyl that may be substituted with -C_1-6alkyl, -C_{1- 6}hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo. In certain embodiments, R is 5- or 6-membered monocyclic heteroaryl that may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo. In certain embodiments, R is pyridinyl that may be substituted with -C_1-6alkyl, -C_1-6hydroxyalky 1, -C_1-6aminoalkyl, -C_1-6haloalky 1, -CN, or -halo. In certain embodiments, R is -C_1-6alkyl that may be substituted with -C_1-6alkyl, -C_1-6hydroxy alkyl, - C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo. In certain embodiments, R is -C_1-6haloalkyl.

In certain embodiments,

. In certain embodiments,

In certain embodiments, ring Y is phenyl that may be substituted with -C_1-6alkyl, -C_{1- 6}hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y are meta to each other. In certain embodiments, ring Y is 6-membered monocyclic heteroaryl that may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_{1- 6}haloalkyl, -CN, or -halo, and the two attachment points on ring Y are meta to each other. In certain embodiments, ring Y is pyridinyl that may be substituted with -C_1-6alkyl, -C_1-6hydroxy alkyl, -C_{1- 6}aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y are meta to each other. In certain embodiments, ring Y is pyridinyl that may be substituted with -C_1-6alkyl, - C_1-6hydroxy alkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y and the nitrogen atom in ring Y are meta to each other.

In certain embodiments,

Z₁ is N;

Z₂ is NR₂; and

Z₃ is N.

In certain embodiments,

Z₁ is CR₁;

Z₂ is NR₂; and

Z₃ is N.

In certain embodiments,

Z₁ is CR₁;

Z₂ is S; and

Z₃ is N.

In certain embodiments,

Z₁ is CR₁;

Z₂ is S; and

Z₃ is CR₃.

In certain embodiments, R₁ is -H or -C_1-6alkyl. In certain embodiments, R₁ is -H.

In certain embodiments, R₂ is -C_1-6alkyl, -C_1-6alkyl-N(C_1-6alkyl)(C_1-6alkyl), or -C_1-6alkyl- O-C_1-6alkyl. In certain embodiments, R₂ is monocyclic cycloalkyl or monocyclic heterocycloalkyl, wherein at least one H of the monocyclic cycloalkyl or monocyclic heterocycloalkyl may be substituted with -C_1-6alkyl, -C_1-6haloalkyl, or -halo.

In certain embodiments, R₃ is -H. In certain embodiments, a compound of the present invention is a compound of Chemical Formula 1 (e.g., a compound of any one of Examples 1 to 60), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. In certain embodiments, a compound of the present invention is a compound of Chemical Formula 1 , or a pharmaceutically acceptable salt thereof.

Use of Heteroaryl Derivative Compound

The present invention provides use of a compound represented by the following Chemical Formula 1 , a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

Chemical Formula 1

According to an embodiment of the present invention, the heteroaryl derivative compound represented by Chemical Formula 1 exhibits excellent inhibitory activity against RAF among kinases, and thus may be usefully employed for the treatment or prevention of RAF-related diseases, in particular, cancer. Specifically, the heteroaryl derivative compound of the present invention exhibits excellent inhibitory activity against RAF mutations and can be usefully employed for treatment or prevention of carcinoma induced by RAF or RAF mutations.

In the present invention, the cancer includes any cancer capable of exhibiting therapeutic or prophylactic efficacy due to inhibition of RAF activity, and may be a solid cancer or a hematologic cancer. For example, the cancer may be one or more selected from the group consisting of pseudomyxoma, intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, labial cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell cancer, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvic cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureter cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoma, gastrointestinal stromal cancer, Wilms cancer, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational choriocarcinoma, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoma, vaginal cancer, spinal cord cancer, acoustic tumor, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymus cancer, but is not limited thereto. The cancer includes not only primary cancer but also metastatic cancer.

In another aspect, the present invention provides a pharmaceutical composition comprising: a compound of the present invention; and optionally a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a kit comprising: a compound or pharmaceutical composition of the present invention; and instructions for using the compound or pharmaceutical composition.

In certain embodiments, the instructions are information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA) or European Medicines Agency (EMA). In certain embodiments, a kit further comprises one or more containers.

According to an embodiment of the present invention, the present invention provides a pharmaceutical composition for treatment or prevention of RAF-related diseases containing the compound represented by Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. Specifically, the RAF-related disease may be cancer. In certain embodiments, the present invention provides a pharmaceutical composition for the treatment or prevention of cancer, comprising the compound, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, as an active ingredient. The types of cancer are the same as described above.

The pharmaceutical composition of the present invention may further include one or more pharmaceutically acceptable carriers in addition to the compound represented by Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof for administration. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, ringer’ s solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and other conventional additives such as antioxidants, buffers, and fungicides can be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added to formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Accordingly, the composition of the present invention may be a patch agent, a liquid agent, a pill, a capsule, a granule, a tablet, a suppository, or the like. These formulations may be prepared by the usual method used in formulation in the art or by the method disclosed in the literature [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and may be formulated into various formulations depending on each disease or ingredient.

The pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar drug efficacy in addition to the compound represented by Chemical Formula 1 above, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound or pharmaceutical composition of the present invention.

In another aspect, the present invention provides a method of preventing a disease in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound or pharmaceutical composition of the present invention.

In certain embodiments, the disease is a RAF-related disease. In certain embodiments, the disease is a disease induced by RAF mutations.

In certain embodiments, the RAF is BRAF.

In certain embodiments, the disease is cancer.

In certain embodiments, the disease is melanoma. In certain embodiments, the disease is colorectal cancer. In certain embodiments, the disease is thyroid cancer. In certain embodiments, the disease is ovarian cancer.

In another aspect, the present invention provides a method of inhibiting the activity and/or production of RAF in a subject, cell, tissue, or biological sample, the method comprising administering to the subject or contacting the cell, tissue, or biological sample with an effective amount of a compound or pharmaceutical composition of the present invention.

In certain embodiments, the subject is a human.

In another aspect, the present invention provides an in vitro method of inhibiting the activity and/or production of RAF in a cell, tissue, or biological sample, the method comprising contacting the cell, tissue, or biological sample with an effective amount of a compound or pharmaceutical composition of the present invention.

Further, according to an embodiment of the present invention, there is provided a method for treating or preventing RAF-related diseases, comprising: administering to a subject in need thereof a therapeutically effective amount of the compound represented by Chemical Formula 1 , a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The subject may be a mammal including a human.

An “effective amount” of a compound or pharmaceutical composition refers to an amount of the compound or pharmaceutical composition sufficient to elicit a desired biological response. An effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics, the condition being treated, the mode of administration, and/or the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount (e.g., when a desired biological response is treatment of a disease). In certain embodiments, the effective amount is a prophylactically effective amount (e.g., when a desired biological response is prevention of a disease).

The term "therapeutically effective amount" as used herein refers to an amount of the compound represented by Chemical Formula 1 that is effective for the treatment or prevention of RAF-related diseases. Specifically, "therapeutically effective amount" indicates an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may be determined depending on factors including the subject type and severity, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, drugs used at the same time, and other factors well-known in medical fields. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. In addition, the pharmaceutical composition of the present invention may be administered in a single dose or multiple doses. It is important to administer the minimum amount capable of obtaining the maximum effect without side effects in consideration of all of the above factors, and the amount may be readily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention may be determined by a medical specialist according to various factors such as the patient's condition, age, sex, complications, and the like. Since the active ingredient of the pharmaceutical composition of the present invention has excellent safety, it may be used at a dose higher than the determined dosage.

Further, according to an embodiment of the present invention, the present invention provides use of the compound represented by Chemical Formula 1, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof for use in preparation of a medicament to treat or prevent RAF-related diseases. The compound represented by Chemical Formula 1 for preparing the medicament may be mixed with acceptable adjuvants, diluents, carriers, and the like, and may have a synergistic effect of active ingredients by being prepared as a complex formulation with other active agents.

Matters mentioned in the uses, compositions and treatment methods of the present invention are applied equally except to the extent that they are inconsistent with each other.

An embodiment of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, embodiments of the present invention are provided to more fully explain the present invention to those with average knowledge in the art. Furthermore, "includes" a component throughout the specification means that it may include other components, not excluding other components unless otherwise opposed.

Advantageous Effects

The heteroaryl derivative compound of the present invention exhibits excellent inhibitory activity against RAF, and thus may be usefully employed for the treatment or prevention of RAF- related diseases.

Best Mode

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely presented to illustrate the present invention, and the scope of the present invention is not limited thereto.

EXAMPLES Compounds synthesized in the Examples of the present invention were purified by the following HPLC conditions or subjected to structural analysis: Medium pressure liquid chromatography (MPLC) for purification; For medium pressure liquid chromatography, CombiFlash Rf+UV from TELEDYNE ISCO was used.

Analytical HPLC Condition (ACQUITY UPLC H-Class Core System)

Waters UPLC system (ACQUITY UPLC PDA Detector) equipped with a mass QDA detector manufactured by Waters was used. The column used was Waters ACQUITY UPLC^® BEH C18 (1.7 2.1 X 50 mm), and the column temperature was performed at 30°C.

Water containing 0.1% formic acid was used as the mobile phase A and acetonitrile containing 0.1% formic acid was used as the mobile phase B.

Gradient conditions (10-100% B over 3 minutes and flow rate = 0.6 ml/min)

Prep-150 LC System for purification (Preparative-Liquid chromatography UV spectrometry)

The equipment manufactured by Waters was used for Prep 150 LC system (2545 Quaternary gradient module, 2998 Photodiode Array Detector, Fraction collector III) manufactured by Waters. The column used was Waters XTERRA^®Prep RP18 OBD™ (10 pm, 30 x 300 mm) and the column temperature was performed at room temperature.

Gradient conditions (3-100% B over 120 minutes and flow rate = 40 ml/min)

The commercial reagent used was used without further refinement.

In the present invention, the room temperature refers to a temperature of about 1 to 35°C.

For concentration under reduced pressure or solvent distillation removal, a rotary evaporator was used.

Preparation Example 1: Preparation of (S)-3-phenylisoxazolidine

Step 1: Preparation of tert-butyl (R)-(3-hydroxy-3-phenylpropoxy)carbamate

Tert-butyl hydroxycarbamate (7.8 g, 58.6 mmol) was dissolved in dimethylformamide (DMF; 140 mL), then NaH(2.58 g, 64.5 mmol) was added at 0°C and the reaction mixture was reacted for 30 min. Next, (R)-3-chloro-l-phenylpropan-l-ol (5 g, 29.3 mmol) dissolved in dimethylformamide (DMF; 10 mL) was slowly added dropwise at 0°C for 10 min, and stirred at room temperature for 72 hours. The reaction was terminated by adding an aqueous ammonium chloride solution to the reaction mixture, followed by extraction with ethyl acetate and brine to combine the organic layers. The organic layer was dried over sodium sulfate and concentrated under reduced pressure, and purified by medium pressure liquid chromatography (ethylacetate/n- hexane) to obtain a target compound (2.8 g, 68%).

MS (m/z): 150.17 [M+l]^{+ 1} H NMR (400 MHz, CDCl₃) δ = 7.43-7.39 (m, 2H), 7.38-7.32 (m, 2H), 7.27-7.24 (m, 1H), 5.05-4.97 (m, 1H), 4.15-4.08 (m, 1H), 4.07-4.00 (m, 1H), 2.10-1.93 (m, 2H), 1.54-1.48 (m, 9H).

Step 2: Preparation of tert-butyl (S)-3-phenylisoxazolidin-2-carboxylate

The compound tert-Butyl (R)-(3-hydroxy-3-phenylpropoxy)carbamate (2.55 g, 9.54 mmol) obtained in Step 1 above and triethylamine (TEA; 3.13 ml, 22.44 mmol) were dissolved in dichloromethane (DCM; 250 ml) and then cooled to 0°C. Next, methanesulfonyl chloride (1 ml, 13 mmol) was added dropwise and stirred at 0°C for 2 hours. The reaction mixture was extracted with brine and dichloromethane, and the organic layers were combined. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the target compound, which was used in the next reaction without purification.

MS (m/z): 194.13 [M+l]^{+ 1} H NMR (400 MHz, CDCI3) δ = 7.29-7.23 (m, 4H), 7.18-7.17 (m, 1H), 5.12-5.11 (m, 1H), 4.10-4.03 (m, 1H), 3.82-3.80 (m, 1H), 2.75-2.65 (m, 1H), 2.29-2.15 (m, 1H), 1.37 (s, 9H).

Step 3: Preparation of (S)-3-phenylisoxazolidine

The compound tert-butyl (S)-3-phenylisoxazolidin-2-carboxylate (2.3 g) obtained in Step 2 was dissolved in dichloromethane (DCM; 90 mL), then trifluoro acetic acid (TFA; 14 mL) was added and the reaction mixture was reacted at room temperature for 1 hour. The reaction mixture was neutralized with an aqueous NaHCO₃ solution, and then the organic layers were combined. The organic layer was dried with sodium sulfate, concentrated under reduced pressure, and purified by medium pressure liquid chromatography (tetrahydrofuran/n-hexane) to obtain a target compound (1.3 g, 94%).

MS (m/z): 150.08 [M+l]^{+ 1}H NMR (400 MHz, DMSO-d₆) δ = 7.59-7.52 (m, 2H), 7.50-7.39 (m, 3H), 5.01-4.93 (m, 1H), 2.93-2.82 (m, 1H), 2.62-2.53 (m, 2H). Preparation Example 2: Preparation of (R)-3-phenylisoxazolidine

A target compound was obtained by synthesizing in the same manner as in Preparation

Example 1, except that (S)-3-chloro-l-phenylpropane-l-ol was used instead of (R)-3-chloro-1- phenylpropane-1-ol in Preparation Example 1.

MS (m/z): 150.08 [M+1] ⁺ Preparation Example 3: Preparation of (R)-3-(3-fluorophenyl)isoxazolidine

Step 1: Preparation of 3-fluoro-N-methoxy-N-methylbenzamide

3-Fluorobenzoic acid (90 g, 642.35 mmol, 1 eq.) was dissolved in pyridine (150 mL), and N-methoxy methanamine (75.19 g, 770.81 mmol, 1.2 eq, HC1) was added thereto. Then, 1-ethyl-

3-(3-dimethylaminopropyl)carbodiimide (EDCI; 147.77 g, 770.81 mmol, 1.2 eq.) was added at 15°C. The reaction mixture was stirred at 50°C for 30 min. As a result of TLC analysis (petroleum ether (PE): ethyl acetate (EA) = 3:1), all of the starting materials disappeared, and a new spot with low polarity was detected. The pyridine solvent was removed by concentration under reduced pressure, and the organic layer was extracted using dichloromethane (DCM; 500 mL), hydrochloric acid (500 mL, 2N), and brine (200 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the target compound as a yellow oil (110 g, 600.50 mmol, 93.49% yield).

1^H NMR (400 MHz, CHLOROFORM-d) δ = 7.47-7.40 (m, 1H), 7.39-7.38 (m, 2H), 7.14- 7.13 (m, 1H), 3.54 (s, 3H), 3.45 (s, 3H).

Step 2: Preparation of 1-(3-fluorophenyl)prop-2-en-1-one

3-Fluoro-N-methoxy-N-methyl-benzamide (110 g, 600.50 mmol, 1 eq.) obtained in Step 1 was dissolved in tetrahydrofuran (THF; 1 L), then bromo(vinyl)magnesium (IM, 630.53 mL, 1.05 eq.) was added dropwise at 0°C. Next, the reaction mixture was stirred at 0°C for 30 min. As a result of TLC analysis (petroleum ether (PE): ethyl acetate (EA) = 4:1), all of the starting materials disappeared, and a new spot with low polarity was detected. The reaction was terminated by adding hydrochloric acid (4N, 500 mL), and the organic layer was extracted using methyl tert-butyl ether (MTBE; 2000 mL) and brine (500 mL). The organic layer was dried over sodium sulfate, followed by concentration under reduced pressure. The concentrated compound was purified by chromatography (petroleum ether(PE)/ethyl acetate(EA) = 30/1) to obtain the target compound as a yellow oil (80 g, 532.80 mmol, 88.73% yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ = 7.65 (m, 1H), 7.58-7.52 (m, 1H), 7.39 (m, 1H), 7.24-7.17 (m, 1H), 7.04 (dd, J= 17.2, 10.4 Hz, 1H), 6.39 (dd, J= 17.2, 1.6 Hz, 1H), 5.90 (dd, J = 10.4, 1.6 Hz, 1H).

Step 3: Preparation of 3-chloro-1-(3-fluorophenyl)propan-1-one 1-(3-Fluorophenyl)prop-2-en-l-one (71 g, 472.86 mmol, 1.0 eq.) obtained in Step 2 was dissolved in dichloromethane (DCM; 71 mL), and HCl/dioxane (4M, 295.54 mL, 2.5 eq.) was added at 0°C. Next, the reaction mixture was stirred at 15°C for 1.5 hours. As a result of TLC analysis (petroleum ether (PE): ethyl acetate (EA) = 10:1), all of the starting materials disappeared, and the target compound was detected. The reaction mixture was concentrated under reduced pressure, and dichloromethane (DCM; 450 mL) and water (200 mL * 5) were added to extract the organic layer. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the target compound as a yellow solid (73 g, 391.19 mmol, 82.73% yield).

¹H NMR (400MHz, CHLOROFORM-d) δ = 7.78-7.72 (m, 1H), 7.69-7.60 (m, 1H), 7.53- 7.44 (m, 1H), 7.37-7.24 (m, 1H), 3.93 (t, 7=6.8 Hz, 2H), 3.46 (t, 7 =6.8 Hz, 2H).

Step 4: Preparation of (S)-3-chloro-1-(3-fluorophenyl)propan-1-ol

(3aR)-1-methyl-3,3-diphenyl-3a,4,5,6-tetrahydropyrrolo[l,2-c][1,3,2]oxazabolol (1M, 32.15 mL, 0.1 eq.) was dissolved in tetrahydrofuran (THF; 1.2L), then borane tetrahydrofuran (BH₃THF; 1M, 186.48 mL, 0.6 eq.) was added dropwise at 0°C under a nitrogen atmosphere. The reaction mixture was stirred at O°C for 30 min. Next, 3-chloro-1-(3-fluorophenyl)propan-1-one (60 g, 309.02 mmol, 1 eq.) obtained in Step 3 diluted in tetrahydrofuran was added dropwise to the reaction mixture at 0°C. The reaction mixture was stirred at 0°C for 30 min. As a result of TLC analysis (petroleum ether (PE): ethyl acetate (EA) = 5:1), all of the starting materials disappeared, and a spot of the target compound was detected. The reaction was terminated by adding methanol (100 mL) at 0°C, and then the solvent was distilled off under reduced pressure. The organic layer was extracted from the concentrated compound using dichloromethane (DCM; 100 mL * 3) and ammonium chloride (NH4CI) solution (300 mL). The organic layer was dried over sodium sulfate, followed by concentration under reduced pressure. The concentrated compound was purified using silica gel chromatography (petroleum ether (PE):ethyl acetate (EA) = 50:1 to 5:1) to obtain the target compound as a colorless oil (140 g, 664.2 mmol, 71.65% yield, 89.49 % purity, 65.5 % e.e).

1^H NMR (400 MHz, CHLOROFORM-d) δ = 7.33 (m, 1H), 7.16-7.07 (m, 2H), 7.02-6.96 (m, 1H), 4.96 (m, 1H), 3.75 (m, 1H), 3.57 (m, 1H), 2.26-2.15(m, 2H).

Step 5: Preparation of tert-butyl (S)-(3-(3-fluorophenyl)-3-hydroxypropoxy)carbamate

Tert-butyl hydroxycarbamate (50.4 g, 378.52 mmol, 1.05 eq.) was dissolved in dimethylformamide (DMF; 500 mL), and then sodium hydride (NaH; 15.86 g, 396.55 mmol, 60% purity, 1.1 eq.) was added at 0°C under a nitrogen atmosphere. The reaction mixture was stirred at 10°C for 1 hour, and (S)-3-chloro-1-(3-fluorophenyl)propan-l-ol (68 g, 360.5 mmol, 1 eq.) obtained in step 4 diluted in dimethylformamide (DMF; 180 mL) was added dropwise at 0°C and stirred at 10°C for 16 hours. As a result of TLC analysis (petroleum ether (PE): ethyl acetate (EA) = 2:1), all of the starting materials disappeared, and the target compound was detected. After the reaction was terminated by adding an aqueous ammonium chloride solution (3L), the organic layer was extracted using ethyl acetate (2000 mL) and brine (2000 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the target compound as a light yellow solid (176 g, 616.87 mmol, 85.56% yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ = 7.67-7.64 (m, 1H), 7.23-7.17 (m, 1H), 7.08- 7.03 (m, 2H), 6.88-6.81 (m, 1H), 4.99-4.84 (m, 1H), 4.02-3.97 (m, 1H), 3.96-3.89 (m, 1H), 1.95- 1.89 (m, 1H), 1.88-1.78 (m, 1H), 1.42-1.39 (m, 9H).

Step 6: Preparation of tert-butyl (R)-3-(3-fluorophenyl)isoxazolidin-2-carboxylate

Tert-butyl (S)-(3-(3-fluorophenyl)-3-hydroxypropoxy)carbamate (88 g, 308.44 mmol, 1 eq.) obtained in step 5 and triethylamine (93.63 g, 925.31 mmol, 128.79 mL, 3 eq.) were dissolved in dichloromethane (DCM; IL), and then methanesulfonic anhydride (80.59 g, 462.65 mmol, 1.5 eq.) was added slowly at 0°C. The reaction mixture was stirred at 20°C for 12 hours. As a result of TLC analysis (petroleum ether (PE): ethyl acetate (EA) = 3:1), all of the starting materials disappeared, and a new spot was detected. After the reaction was terminated by adding water (2000 mL), the organic layer was extracted using dichloromethane (DCM; 200 mL * 3). The organic layer was dried over sodium sulfate, followed by concentration under reduced pressure. The concentrated compound was purified by chromatography (petroleum ether (PE):ethyl acetate (EA) = 50:1 to 5:1) to extract 88 g of the target compound having an 82.5% e.e value. The target compound was purified through SFC (column: DAICEL CHIRALPAK AD (250mm * 50mm, 10μm); mobile phase: [Neu-MeOH]; B%: 15%-15%, 3.4min; 380min) to obtain the target compound as a white solid (51 g, 189.66 mmol, 30.74% yield, 99.4% purity).

The purity of the stereoisomer of tert-butyl (R)-3-(3-fluorophenyl)isoxazolidin-2- carboxylate obtained in step 6 above was analyzed under SFC conditions as follows.

Instrument: CAS-WH-ANA-SFC-C (SHIMADZU LC-30ADsf)

Column: Amycoat 50x4.6mm I.D., 3um

Mobile phase: Phase A for CO₂, and Phase B for MeOH(0.05% DEA);

Gradient elution: MeOH(0.05% DEA) in CO₂ from 5% to 40%

Flow rate: 3mL/min; Detector: PDA;

Column Temp: 35°C; Back Pressure: 100 Bar

When the purity of the stereoisomer of tert-butyl (R)-3-(3-fluorophenyl)isoxazolidin-2- carboxylate obtained in step 6 was low, the desired stereoisomer as a yellow liquid was obtained by performing purification under SFC conditions as follows.

Column: DAICEL CHIRALPAK AD-H (250mm * 30mm, 5um);

Mobile phase: [0.1% NH₃H₂O MeOH]; B%: 15%-15%, 3.8min; 600min

Step 7: Preparation of (R)-3-(3-fluorophenyl)isoxazolidine

Tert-butyl (R)-3-(3-fluorophenyl)isoxazolidin-2-carboxylate (50 g, 185.94 mmol, 1 eq.) obtained in step 6 was dissolved in ethyl acetate (EA; 200 mL), and then HCl/EtOAc (4M, 300 mL, 6.45 eq.) was added at 0°C. Next, the reaction mixture was stirred at 10°C for 1 hour. As a result of LCMS analysis, all of the starting materials disappeared and the reaction mixture was concentrated under reduced pressure to obtain a solid to thereby yield the target compound as a white solid (32 g, 150.26 mmol, 80.81% yield, 95.62 % purity, 100 % e.e. HC1). MS: m/z 168.2 [M+H]^{+ 1} H NMR (400 MHz, DMSO-d₆) δ = 7.53-7.43 (m, 2H), 7.39 (d, 7 = 7.8 Hz, 1H), 7.30-7.23 (m, 1H), 5.01 (t, J = 8.0 Hz, 1H), 4.47 (m, 1H), 4.27 (m, 1H), 2.87 (m, 1H), 2.62-2.52 (m, 1H)

The following conditions were employed for purification or analysis of stereoisomers of the compound in step 7 above.

Instrument: CAS-WH-ANA-SFC-C(SHIMADZU LC-30ADsf) Column: Chiralpak AY-3 50x4.6mm I.D., 3um;

Mobile phase: Phase A for CO₂, and Phase B for IPA(0.05% DEA); Gradient elution: B in A from 5% to 40%;

Flow rate: 3mL/min; Detector: PDA; Column Temp: 35°C; Back Pressure: 100 Bar

Preparation Examples 4 to 16 The following compounds of Preparation Examples 4 to 16 were prepared in a manner similar to those of Preparation Examples 1 to 3 above, and the names of compounds and chemical structural formulas of Preparation Examples 4 to 16 are summarized in Table 1.

Table 1

Example 1: Preparation of (S)-N-(3-((4-amino-l-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)phenyl)-3-phenylisoxazolidin-2-carboxamide

Step 1: Preparation of 1-ethyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-amine(5 g), iodoethane(3.14 g, 1.05 eq), and K₂CO₃(5.29 g, 2 eq) were added to dimethylformamide (DMF; 38 ml) and stirred at 80°C overnight. Thereafter, the reaction mixture was cooled to room temperature, and water was added, and the resultant solid was filtered and recovered. The filtered solid was dried to obtain a target compound (3.5 g, 63% yield).

MS (m/z): 290.0 [M+H] ⁺

Step 2: Preparation of (S)-N-(3-ethynylphenyl)-3-phenylisoxazolidine-2-carboxamide 3-ethynylaniline(35 g) and pyridine(47.3 g, 2 eq) were dissolved in dichloromethane(DCM; 350 ml). Phenylchloroformate(51.5 g, 1.1 eq) was slowly added dropwise at 0°C and stirred at room temperature for 2 hours. After confirming that the reaction substrate was completely consumed, (S)-3-phenylisoxazolidine(53.5 g, 1.2 eq) obatained from Preparation Example 1 and triethylamine( 116 g, 3 eq) were added and stirred at 45°C for 12 hours. The reaction mixture was cooled to room temperature, and an organic layer was extracted using water(200 mL x 2), brine(200 mL), and dichloromethane (DCM)(200 ml x 2). The organic layer was dried with sodium sulfate and then concentrated under reduced pressure. It was purified using medium- pressure liquid chromatography(ethylacetate/n-hexane) to obtain a target compound (69 g, 98% yield).

MS (m/z): 293.2 [M+H] ⁺

Step 3: Preparation of (S)-N-(3-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidine-3- yl)ethynyl)phenyl)-3-phenyl isoxazolidine-2-carboxamide

1-ethyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-amine (100 mg) obtained in step 1, (S)- N-(3-ethynylphenyl)-3-phenylisoxazolidine-2-carboxamide(126 mg, 1.25 eq) obtained in step 2, triethylamine(TEA; 0.145 ml), Cul(3 mg), Pd(PPh₃)₂Cl₂(25 mg) were dissolved in dimethylformamide(DMF; 3 ml) and stirred at 70 °C for 1 hour. After that, the reaction mixture was cooled to room temperature, brine was added, and an organic layer was extracted using ethylacetate(EA). The organic layer was dried with sodium sulfate and then concentrated under reduced pressure. It was purified with Prep- 150 LC System to obtain a target compound(110 mg, 70% yield).

MS (m/z): 454.2 [M+H] ⁺ Preparation Examples 2 to 60

The compounds of Examples 2 to 60 were prepared in a manner similar to Preparation Example 1 using Preparation Examples 1 to 16.

The compound name, chemical structural formula, NMR, and LC-MS analysis results of Examples 1 to 60 are summarized in Table 2 below.

Table 2

Z1

Experimental Example 1: Evaluation of BRAF, BRAF(V600E), RAFI inhibitory activity of compounds according to the present invention The following experiments were performed to evaluate the BRAF, BRAF(V600E), and

RAF1 inhibitory activity of the compounds according to the present invention. Specifically, for the compound of Example 4 selected from among the compounds of the present invention, the enzyme(kinase) selectivity was measured by requesting DiscoverX, and the experiment was conducted using the scanMAX™ Kinase analysis panel. At this time, the concentration of the drug treated with the enzyme was 1 μM in DMSO, and the control percentage(% control) was determined in the same way as in Formula 1 below, and the results are shown in Table 3 below. Formula 1 (Example Compound - Positive Control)/(negative Control - Positive Control) x 100

Here, the positive control refers to a compound representing a control percentage of 0%, and the negative control is DMSO, indicating a control percentage of 100%. In addition, the enzyme selectivity of the present invention was determined to have activity for the enzyme when the control percentage for the enzyme is <35% (ie, less than 35%).

Table 3

As shown in Table 3, the Example compound according to the present invention has a value less than 35% of the control percentage for BRAF, BRAF (V600E), and RAFI, so that it can be seen that it has activity against BRAF, BRAF (V600E), and RAFI. This suggests that the Example compound according to the present invention has a useful effect when used for BRAF, BRAF (V600E), and RAF1-related diseases, and thus may be useful as a composition for treating or preventing BRAF, BRAF (V600E), and RAF1-related diseases.

Experimental Example 2: Evaluation of cell proliferation inhibition activity in A375P cell of the compound according to the present invention

The following experiments were performed to evaluate the cell proliferation inhibitory activity of the compounds according to the present invention. Cell viability analysis was performed by culturing A375P cell line(Korea Cell Line Bank #80003) with a medium of Dulbecco's Modified Eagles Medium(High Glucose)(Hyclone #SH30243.01) containing 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin. More specifically, when performing the test, the cell line was aliquoted in a 96- well flat-bottom plate(corning #3903) at a concentration of 3,000 cells/well, respectively, and then cultured at 37°C for 24 hours under 5% CO₂ conditions. Compounds in each well were treated with 11 concentrations by giving a 3 -fold concentration gradient to the highest concentration of 10 μM, and dimethylsulfoxide(DMSO) was treated at the same concentration of 0.5% (v/v) as in the compound treatment as each control group. The compound treated cells were incubated for 72 hours. To check the degree of cell viability, 100 μl of Cell Titer-Glo(Promega #G7573) was added to the culture medium of each cultured cell, and then incubated for another 10 minutes at room temperature, and then luminescence was measured using a microplate reader. The degree of cell proliferation inhibitory activity according to the treatment concentration of each compound was calculated based on the luminescence of the control cells not treated with the compound, and the concentration with 50% cell proliferation inhibitory activity was determined as the GI₅₀(uM) value. GI₅₀(uM) value was obtained using Prism(version 8.4.3 #GraphPad) software, and the results are shown in Table 4.

Experimental Example 3: Evaluation of cell proliferation inhibition activity in HCT116 cell of the compound according to the present invention

The following experiments were performed to evaluate the cell proliferation inhibitory activity of the compounds according to the present invention. Cell viability analysis was performed by culturing HCT116 cell line(Korea Cell Line Bank #10247) with a medium of McCoy's 5A (Modified) Medium(Gibco #16600082) containing 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin. More specifically, when performing the test, the cell line was aliquoted in a 96-well flat-bottom plate(corning #3903) at a concentration of 2,000 cells/well, respectively, and then cultured at 37 °C for 24 hours under 5% CO₂ conditions. Compounds in each well were treated with 11 concentrations by giving a 3 -fold concentration gradient to the highest concentration of 10 μM, and dimethylsulfoxide(DMSO) was treated at the same concentration of 0.5% (v/v) as in the compound treatment as each control group. The compound treated cells were incubated for 72 hours. To check the degree of cell viability, 100 pl of Cell Titer-Glo(Promega #G7573) was added to the culture medium of each cultured cell, and then incubated for another 10 minutes at room temperature, and then luminescence was measured using a microplate reader. The degree of cell proliferation inhibitory activity according to the treatment concentration of each compound was calculated based on the luminescence of the control cells not treated with the compound, and the concentration with 50% cell proliferation inhibitory activity was determined as the GI₅₀(uM) value. GI₅₀(uM) value was obtained using Prism(version 8.4.3 #GraphPad) software, and the results are shown in Table 4.

Table 4

Claims

CLAIMS What is claimed is:

1. A compound represented by the following Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

(Chemical Formula 1) in the Chemical Formula 1 ,

X is CH₂ or O;

R is -C_1-6alkyl, -C_1-6haloalkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl in which at least one H of the aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, - C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo; ring Y is aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl in which at least one H of the aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_{1 6}haloalkyl, -CN, or -halo;

Z₁ is CR₁, NR₂, or N;

Z₂ is NR₂, N, O, or S;

Z₃ is CR₃ or N;

R₁ and R₂ are each independently -H, -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_{1 6}haloalkyl, -C_1-6alkyl-N(C_1-6alkyl)(C_1-6alkyl), -C_1-6alkyl-O-C_1-6alkyl, cycloalkyl or heterocycloalkyl, wherein at least one H of the cycloalkyl or heterocycloalkyl may be substituted with -C_1-6alkyl, -C_1-6haloalkyl, or -halo; and

R₃ is -H or -C_1-6alkyl.

2. The compound of claim 1, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein

X is CH₂ or O;

R is -C_1-6alkyl, -C_1-6haloalkyl, aryl, or heteroaryl, wherein at least one H of the aryl or heteroaryl may be substituted with -C_1-6alkyl, -C_1-6haloalkyl, -CN, or -halo; a ring Y is aryl or heteroaryl, wherein at least one H of the aryl or heteroaryl may be substituted with -C_1-6alkyl or -halo;

Z₁ is CR₁, NR₂, or N;

Z₂ is NR₂, N, or S;

Z₃ is CR₃ or N;

Ri and R₂ are each independently -H, -C_1-6alkyl, -C_1-6alkyl-N(C_1-6alkyl)(C_1-6alkyl), -C_{1- 6}alkyl-O-C_1-6alkyl, 3 to 7 membered cycloalkyl or 3 to 7 membered heterocycloalkyl in which at least one H of the 3 to 7 membered cycloalkyl or 3 to 7 membered heterocycloalkyl may be substituted with -C_1-6alkyl or -halo; and

R₃ is -H.

3. The compound of any one of claims 1-2, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein X is O.

4. The compound of any one of claims 1-2, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein X is CH₂.

5. The compound of any one of claims 1-4, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R is phenyl that may be substituted with -C_{1- 6}alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo.

6. The compound of any one of claims 1-4, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R is 5- or 6-membered monocyclic heteroaryl that may be substituted with -C_1-6alkyl, -C_1-6hydroxy alkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo.

7. The compound of any one of claims 1-4, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R is pyridinyl that may be substituted with -C_{1- 6}alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo.

8. The compound of any one of claims 1-4, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R is -C_1-6alkyl that may be substituted with -C_{1 6}alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo.

9. The compound of any one of claims 1-4, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R is -C_1-6haloalkyl.

10. The compound of any one of claims 1-9, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein

11. The compound of any one of claims 1-9, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein

12. The compound of any one of claims 1-9, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein

13. The compound of any one of claims 1-12, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein ring Y is phenyl that may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y are meta to each other.

14. The compound of any one of claims 1-12, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein ring Y is 6-membered monocyclic heteroaryl that may be substituted with -C_1-6alkyl, -C_1-6hydroxy alkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y are meta to each other.

15. The compound of any one of claims 1-12, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein ring Y is pyridinyl that may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y are meta to each other.

16. The compound of any one of claims 1-12, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein ring Y is pyridinyl that may be substituted with -C_1-6alkyl, -C_1-6hydroxyalkyl, -C_1-6aminoalkyl, -C_1-6haloalkyl, -CN, or -halo, and the two attachment points on ring Y and the nitrogen atom in ring Y are meta to each other.

17. The compound of any one of claims 1-16, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein:

Z₁ is N;

Z₂ is NR₂; and

Z₃ is N.

18. The compound of any one of claims 1-16, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein:

Z₁ is CR₁;

Z₂ is NR₂; and

Z₃ is N .

19. The compound of any one of claims 1-16, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein:

Z₁ is CR₁;

Z₂ is S; and

Z₃ is N .

The compound of any one of claims 1-16, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein:

Z₁ is CR₁;

Z₂ is S; and

Z₃ is CR₃.

21. The compound of any one of claims 1-20, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R₁ is -H or -C_1-6alkyl.

22. The compound of any one of claims 1-20, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R₁ is -H.

23. The compound of any one of claims 1-22, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R₂ is -C_1-6alkyl, -C_1-6alkyl-N(C_1-6alkyl)(C_{1- 6}alkyl), or -C_1-6alkyl-O-C_1-6alkyl.

24. The compound of any one of claims 1-22, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R₂ is monocyclic cycloalkyl or monocyclic heterocycloalkyl, wherein at least one H of the monocyclic cycloalkyl or monocyclic heterocycloalkyl may be substituted with -C_1-6alkyl, -C_1-6haloalky 1, or -halo.

25. The compound of any one of claims 1-24, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein R₃ is -H.

26. The compound of claim 1, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, wherein the compound represented by the Chemical Formula 1 is selected from the group consisting of the following compounds:

(1) (S)-N-(3-((4-amino-l-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenyl)-3- phenylisoxazolidin-2-carboxamide;

(2) (S)-N-(3-((4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethynyl)phenyl)-3- phenylisoxazolidin-2-carboxamide;

(3) (S)-N-(3-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)phenyl)-3-phenylisoxazolidin-2- carboxamide; (4) (R)-N-(3-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- phenylisoxazolidin-2-carboxamide;

(5) (R)-N-(3-((4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethynyl)-4-methylphenyl)-3- phenylisoxazolidin-2-carboxamide;

(6) (R)-N-(3-((4-amino-2-isopropyl-2H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(7) (R)-N-(3-((4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(8) (R)-N-(5-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(9) (R)-N-(5-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-6-methylpyridin-3- yl)-3-phenylisoxazolidin-2-carboxamide;

(10) (R)-N-(3-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)-4-methylphenyl)-3- phenylisoxazolidin-2-carboxamide;

(11) (R)-N-(3-((4-amino-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(12) (R)-N-(3-((4-amino-1-(2-methoxyethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(13) (R)-N-(3-((4-amino-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(14) (R)-N-(3-((4-amino-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(15) (R)-N-(5-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-bromo-2- fluorophenyl)-3-phenylisoxazolidin-2-carboxamide;

(16) (R)-N-(3-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3-chloro-4-fluorophenyl)isoxazolidin-2-carboxamide;

(17) (R)-N-(3-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (4-chloro-3-fluorophenyl)isoxazolidin-2-carboxamide;

(18) (R)-N-(3-((4-amino-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(19) (R)-N-(3-((4-amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)- 4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(20) (R)-N-(5-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2-fluorophenyl)-3- phenylisoxazolidin-2-carboxamide;

(21) (R)-N-(5-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2-fluoropyridin-3- yl)-3-phenylisoxazolidin-2-carboxamide;

(22) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3-fluorophenyl)isoxazolidin-2-carboxamide;

(23) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (2,5-difluorophenyl)isoxazolidin-2-carboxamide;

(24) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3-chlorophenyl)isoxazolidin-2-carboxamide;

(25) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3-cyanophenyl)isoxazolidin-2-carboxamide;

(26) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (pyridin-3-yl)isoxazolidin-2-carboxamide;

(27) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (4-fluorophenyl)isoxazolidin-2-carboxamide;

(28) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3,5-difluorophenyl)isoxazolidin-2-carboxamide;

(29) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (2,3-difluorophenyl)isoxazolidin-2-carboxamide;

(30) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3-fluoro-5-(trifluoromethyl)phenyl)isoxazolidin-2-carboxamide;

(31) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (4-chlorophenyl)isoxazolidin-2-carboxamide;

(32) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3- (3-chloro-2-fluorophenyl)isoxazolidin-2-carboxamide;

(33) (R)-N-(3-((4-amino- 1-methyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)- 3-phenylisoxazolidin-2-carboxamide;

(34) (R)-N-(3-((4-amino- 1-ethyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2, 4- difluorophenyl)-3-phenylisoxazolidin-2-carboxamide;

(35) (R)-N-(3-((4-aminothieno[3,2-c]pyridin-3-yl)ethynyl)-4-methylphenyl)-3- phenylisoxazolidin-2-carboxamide;

(36) (R)-N-(3-((4-amino-1-((S)-1-methylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(37) (R)-N-(3-((4-amino-1-((S)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(38) (R)-N-(3-((4-amino-1-((3S,4S)-3-fluoro-1-methylpiperidin-4-yl)-1H-pyrazolo[3,4- d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(39) (R)-N-(3-((4-amino-1-(2-(dimethylamino)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(40) (R)-N-(3-((4-amino-1-(3-(dimethylamino)propyl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(41 ) (R)-N -(3 -((4-amino- 1 -((R)-pyrrolidin-3 -yl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-3 -yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(42) (R)-N-(3-((4-amino-1-(azetidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(43 ) (R)-N -(3 -((4-amino- 1 -( 1 -methylazetidin-3 -yl)- IH-pyrazolo [3 ,4-d]pyrimidin-3 -yl)ethynyl)- 4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(44) (R)-N-(3-((4-amino-1-((R)-1-methylpyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3- yl)ethynyl)-4-methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(45) (R)-N-(5-((4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-(3-fluorophenyl)isoxazolidin-2-carboxamide;

(46) (R)-N-(5-((4-amino-l-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-(3,5-difluorophenyl)isoxazolidin-2-carboxamide;

(47) (R)-N-(5-((4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethynyl)-6-methylpyridin-3- yl)-3-phenylisoxazolidin-2-carboxamide;

(48) (R)-N-(5-((4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-phenylisoxazolidin-2-carboxamide;

(49) (R)-N-(5-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)-6-methylpyridin-3-yl)-3- phenylisoxazolidin-2-carboxamide;

(50) (R)-N-(5-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)-2-fluoro-4-methylphenyl)-3- phenylisoxazolidin-2-carboxamide;

(51) (R)-N-(3-((4-amino-l-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)- 3-(3-fluorophenyl)isoxazolidin-2-carboxamide;

(52) (R)-N-(3-((4-amino- 1-methyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)- 3-(3,5-difluorophenyl)isoxazolidin-2-carboxamide;

(53) (R)-N-(5-((4-amino- 1-methyl- 1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-(3-fluorophenyl)isoxazolidin-2-carboxamide;

(54) (R)-N-(5-((4-amino- 1-methyl- 1H-pyrazolo[3, 4-d]pyrimidin-3-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-(3,5-difluorophenyl)isoxazolidin-2-carboxamide;

(55) (R)-N-(5-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)-2-fluoro-4-methylphenyl)-3-(3- fluorophenyl)isoxazolidin-2-carboxamide;

(56) (R)-N-(5-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)-2-fluoro-4-methylphenyl)-3-(3,5- difluorophenyl)isoxazolidin-2-carboxamide;

(57) (R)-N-(5-((4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-(3-fluorophenyl)isoxazolidin-2-carboxamide;

(58) (R)-N-(5-((4-amino-7-ethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethynyl)-2-fluoro-4- methylphenyl)-3-(3,5-difluorophenyl)isoxazolidin-2-carboxamide;

(59) N-(3-((4-amino-l-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-4-methylphenyl)-3-

(2,2,2-trifluoroethyl)pyrrolidin- 1-carboxamide; and

(60) N-(3-((4-aminothieno[2,3-d]pyrimidin-5-yl)ethynyl)-4-methylphenyl)-3-(2,2,2- trifluoroethyl)pyrrolidin- 1 -carboxamide.

27. The compound of any one of claims 1-26, or the pharmaceutically acceptable salt thereof.

28. A pharmaceutical composition comprising: a compound of any one of claims 1-26, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof; and optionally a pharmaceutically acceptable excipient.

29. The pharmaceutical composition of claim 28 further comprising one or more active ingredients.

30. A kit comprising: a compound of any one of claims 1-26, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 28-29; and instructions for using the compound, stereoisomer, pharmaceutically acceptable salt, or pharmaceutical composition.

31. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound of any one of claims 1-26, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 28-29.

32. A method of preventing a disease in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a compound of any one of claims 1-26, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 28-29.

33. The method of any one of claims 31-32, wherein the disease is a RAF-related disease.

34. The method of any one of claims 31-33, wherein the disease is a disease induced by RAF mutations.

35. The method of any one of claims 33-34, wherein the RAF is BRAF.

36. The method of any one of claims 31-35, wherein the disease is cancer.

37. The method of any one of claims 31-35, wherein the disease is melanoma.

38. The method of any one of claims 31-35, wherein the disease is colorectal cancer.

39. The method of any one of claims 31-35, wherein the disease is thyroid cancer.

40. The method of any one of claims 31-35, wherein the disease is ovarian cancer.

41. A method of inhibiting the activity and/or production of RAF in a subject, cell, tissue, or biological sample, the method comprising administering to the subject or contacting the cell, tissue, or biological sample with an effective amount of a compound of any one of claims 1-26, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 28-29. 42. The method of any one of claims 31-41, wherein the subject is a human. 43. An in vitro method of inhibiting the activity and/or production of RAF in a cell, tissue, or biological sample, the method comprising contacting the cell, tissue, or biological sample with an effective amount of a compound of any one of claims 1-26, or the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 28- 29. 44. The method of any one of claims 41-43, wherein the RAF is BRAF. 45. A pharmaceutical composition for the treatment or prevention of cancer, comprising the compound, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 26 as an active ingredient.