Compounds modulating c-kit activity and uses therefor

Compound for regulating C-KIT activity and its use

The present invention relates to the ligands of c-kit and the use of the ligands. The information provided is only intended to help readers understand. Neither the information provided nor the references cited are regarded as prior art of the present invention. The full text of each cited reference is incorporated into this article.

The receptor protein tyrosine kinase (RPTK) regulates the key signal transduction cascade that controls cell growth and proliferation. Stem cell factor (SCF) receptor c-kit is a type III transmembrane RPTK, which includes five extracellular immunoglobulin (IG) domains, a single transmembrane domain, and a lytic cytoplasmic kinase domain separated by a kinase insert. C-kit plays an important role in the growth of melanocytes, mast cells, germ cells and hematopoietic cells.

Stem cell factor (SCF) is a protein encoded by the S1 block, and based on the biological characteristics used to identify the SCF, it has been called kit ligand (KL) and mast cell growth factor (MGF) (in Tsujimura,<i>Pathol Int</i>1996,<b>46</b>: 933-938; Loveland et al.,<i>J. Endocrinol</i>1997,<b>153</b>: 337-344; Vliagoftis et al.,<i>Clin Immunol</i>1997,<i>100</i>: 435-440; Broudy,<i>Blood</i>1997,<b>90</b>:1345-1364; Pignon,<i>Hermatol Cell Ther</i>1997,<b>39</b>: 114-116; and Lyman et al., Blood 1998, 91:1101-1134. ). In this article, we use the abbreviation SCF to refer to the ligand of c-kit RTK.

SCF can be synthesized as a transmembrane protein with a molecular weight of 220 or 248 Daltons, depending on the alternative splicing of the mRNA encoding exon 6. Larger proteins can be cleaved by proteolysis to form soluble glycosylated proteins with non-covalent dimerization. Both soluble and membrane-bound forms of SCF can bind to c-kit and activate c-kit. For example, in the skin, SCF is mainly expressed by fibroblasts, keratinocytes, and endothelial cells that regulate the activity of melanocytes and mast cells that express c-kit. In bones, myeloid stromal cells express SCF and regulate the blood cell production of c-kit, which expresses stem cells. In the gastrointestinal tract, intestinal epithelial cells express SCF and affect interstitial cells of Cajal and intraepithelial lymphocytes. In the testis, sertoli cells and granulosa cells express SCF, which regulate sperm production by interacting with c-kit on germ cells.

Additional RPTK proteins such as Ret and NTRK1 have been described (Takahashi &amp; Cooper, Mol Cell Biol. 1987, 7:1378-85; Bothwell, Cell. 1991, 65:915-8). Ret and NTRK1 play a role in the development and maturation of specific components of the nervous system. The changes in Ret and NTRK1 have been linked to several human diseases, including certain forms of cancer and dysplasia. The correlation between genetic changes and the appearance of various diseases has helped to form the idea that one gene can be responsible for more than one disease. In addition, gene changes in Ret and NTRK1 belonging to the mutation category of "increased function" or "loss of function" have been observed. In fact, receptor rearrangements or point mutations convert Ret and NTRK1 into the main functional transformation genes that cause thyroid tumors, which are associated with Hirschsprung's disease (HSCR) and congenital hyperalgesia combined with anhidrosis Non-activating mutations related to CIPA damage the functions of Ret and NTRK1, respectively.

Abnormal manifestations and/or activation of c-kit have been implied in many pathologies. For example, evidence that c-kit contributes to tumor pathology includes its association with leukemia and mast cell tumors, small cell lung cancer, testicular cancer, and certain gastrointestinal and central nervous system cancers. In addition, it has been suggested that c-kit plays a role in the carcinogenesis of female reproductive tract, neuroectodermal sarcoma and Schwann cell tumor formation related to multiple neurofibromas. We have found that mast cells are involved in altering the tumor microenvironment and enhancing tumor growth (Yang et al.,<i>J Clin Invest.</i>2003,<b>112</b>: 1851-1861; Viskochil,<i>J Clin Invest.</i>2003,<b>112</b>: 1791-1793). Therefore, a regulator of c-kit activity is needed in this technology.

The present invention relates to compounds having activity on c-kit. In detail, the present invention provides the compound of formula I as described below. Therefore, the present invention provides compounds that can be used in therapeutic and/or preventive methods involving the regulation of c-kit.

The compound of formula I has the following structure:<chemistry general="n"><img file="TW200616632A_D0001.tif" /></chemistry>

Wherein Z is halogen or optionally methyl substituted by halogen.

With regard to compounds of formula I, the following definitions apply.

"Halo" or "halogen" alone or in combination means all halogens, including chlorine (Cl), fluorine (F), bromine (Br), and iodine (I).

"Methyl" alone or in combination means having the structure -CH₃ofalkyl. "Methyl substituted with halogen" refers to a methyl substituted with 1 or more (for example, 1, 2 or 3) halogens, such as -CH₂Cl, -CF₃And its analogues.

"Alkyl" alone or in combination means a group derived from an alkane containing 1 to 20, preferably 1 to 15 carbon atoms. Alkyl groups include straight chain alkyl groups and branched chain alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, tertiary butyl, and the like. The straight or branched chain alkyl group contains 1 to 15, more preferably 1 to 8, even more preferably 1 to 6, even more preferably 1 to 4, and most preferably 1 to 2 carbon atoms. Straight or branched chain alkyl groups are attached at any available point to produce stable compounds.

"Substituted alkyl" is an alkyl group independently substituted with 1 or more (for example, 1, 2 or 3) groups or substituents (such as halo or the like).

Regarding formula I, the unsubstituted core pyrrole[2,3-b]pyridine structure shown above is called the "azainimid core". For the azaindole core, the reference system of ring atoms or ring positions is shown in the following structure:<chemistry general="n"><img file="TW200616632A_D0002.tif" /></chemistry>

Regarding the c-kit modulator compound herein, the description of the compound or compound group includes the pharmaceutically acceptable salts of the compound(s), and unless explicitly stated to the contrary, it also includes prodrugs and all isomers . The term "prodrug" as used herein refers to a compound that produces the desired active compound when metabolized. Prodrugs generally have no activity or are less active than the active compound, but they can provide advantageous handling, administration, or metabolic properties. For example, certain prodrugs are esters of the active compound; during metabolism, the ester group is cleaved to generate the active drug. In addition, certain prodrugs are activated by enzymatic action to generate active compounds or compounds that are further chemically reacted to generate active compounds.

Therefore, in the first aspect, the present invention provides for the treatment of diseases or disorders regulated by c-kit in animal subjects (e.g., mammals, such as humans) (e.g., with abnormal c-kit activity (e.g., kinase activity) as Characteristic disease or disorder) method, wherein the method comprises administering an effective amount of a compound of formula I to the subject.

As used herein, the term "disease or condition regulated by c-kit" refers to a disease or condition in which the biological function of c-kit influences the progression and/or process of a disease or condition and/or the regulation of c-kit changes the disease or condition A disease or condition that progresses, processes, and/or symptoms. For example, mutations in the c-kit gene (such as the W42, Wv, and W41 mutations reported by Herbst et al. (J. Biol. Chem., 1992, 267: 13210-13216)) confer severe, intermediate, and mild mutations, respectively. Phenotypic characteristics. These mutations weaken the inherent tyrosine kinase activity of the receptor to varying degrees, and it is a model of the regulatory effect of c-kit activity.

Exemplary diseases or conditions that can be treated or prevented include (but are not limited to) cancer, asthma, arthritis, chronic rhinitis, multiple sclerosis, GIST, and mastocytosis.

In a related aspect, the compound of formula I can be used to prepare drugs for the treatment of diseases or disorders (such as cancer, asthma, arthritis, chronic rhinitis, multiple sclerosis or other diseases) modulated by c-kit.

In another aspect, the present invention provides compounds as described herein (e.g., compounds having a favorable degree of activity and/or selectivity to c-kit).

In a specific embodiment, the compound has an IC of less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, or less than 5 nM as determined in generally accepted kinase activity assays₅₀. In certain embodiments, the selectivity of the compound is such that the compound has at least 2-fold, 5-fold, 10-fold, or 100-fold more activity against c-kit than c-ret. In certain embodiments, the compound has the activity specified in this paragraph (e.g., IC₅₀) And/or optional.

An additional aspect of the present invention relates to a composition, which includes a therapeutically effective amount of a compound of formula I (or a compound in any subgroup of compounds within the general formula) and at least one pharmaceutically acceptable carrier, excipient, and / Or thinner. The composition may include a plurality of different pharmacologically active compounds, which may include a plurality of compounds of formula I.

As used herein, the term "composition" refers to a formulation suitable for administration to a predetermined animal subject for therapeutic purposes, which contains at least one pharmaceutically active compound and at least one pharmaceutically acceptable carrier or excipient Shape agent.

The term "pharmaceutically acceptable" means that the material in question does not have the characteristics of urging a moderately cautious physician to consider the disease or condition to be treated and the respective route of administration and avoid administering the material to the patient. For example, it is generally required that the material be substantially sterile, such as in the case of injectables.

In the context of the present invention, the term "therapeutically effective" or "effective amount" means that the material or the amount of the material can effectively prevent, reduce or ameliorate the symptoms of one or more diseases or medical conditions and/or prolong the treatment The life span of the subject.

In a related aspect, the present invention provides kits comprising the composition as described herein. In a specific embodiment, the composition is packaged in (for example) vials, bottles, flasks, and the container may be further packaged in (for example) boxes, envelopes or bags; the composition is approved by the US Food and Drug Administration Bureau or similar regulatory agency approved for administration to mammals (such as humans); the composition is approved for administration to mammals (such as humans) for diseases or disorders regulated by c-kit; the kit of the present invention includes Written instructions and/or other indications that the composition is suitable or approved for administration to mammals (such as humans) for diseases or conditions regulated by c-kit; and the combination is packaged in unit dose or single dose form Such as single-dose pills, capsules or the like.

In aspects including treatment or prevention of a disease or condition, the disease or condition is cancer, asthma, arthritis, chronic rhinitis, multiple sclerosis, mastocytosis or other diseases.

In certain embodiments, the c-kit modulator has a serum half-life of longer than 2 hours, longer than 4 hours, or longer than 8 hours; water solubility; oral bioavailability of greater than 10% or oral bioavailability of greater than 20%.

The reference of specific amino acid residues in the number of human c-kit polypeptide residues is defined by numbering the sequence corresponding to GenBank NP_000213 (sequence number: 1). By numbering corresponding to the sequence provided by GenBank NM_000222 (serial number: 2), the reference of the specific nucleotide position in the nucleotide sequence encoding all or part of c-kit is defined.

The term "c-kit" means an enzymatically active kinase, which contains amino acid residues greater than the ATP binding site including full-length c-kit (such as human c-kit, such as sequence NP_000213, sequence number: 1) 90% amino acid sequence identity part for maximum alignment by equal length fragments; or contains at least 200 adjacent amino acids with natural c-kit with greater than 90% amino acid sequence identity Sexual part and maintain kinase activity. The sequence identity is preferably at least 95, 97, 98, 99, 99.9% or 100%. The specific level of sequence identity is preferably on a sequence of at least 300 adjacent amino acid residues in length. Unless stated to the contrary, the terms "kit" and "c-kit" include references to wild-type c-kit, allele variants, and mutant forms (for example, with activating mutations). The term "c-kit activity" refers to the biological activity of c-kit, especially including kinase activity.

The term "c-kit kinase domain" refers to a truncated c-kit that includes the kinase catalytic region of c-kit (ie, shorter than the full-length c-kit by at least 100, at least 200, at least 300, or more than 300 amino acids ). Ideally, for use in the present invention, the kinase domain retains kinase activity, preferably at least 60, 70, 80, 90 or 100% of the natural c-kit kinase activity.

As used herein, the terms "ligand" and "modulator" are equivalently used to refer to compounds that alter (ie increase or decrease) the activity of a target biomolecule (for example, an enzyme, such as a kinase). Ligands or modifiers will generally be small molecules, where "small molecules" refer to their molecular weights of 1500 Daltons or less, or preferably 1000 Daltons or less, 800 Daltons or less, Or compounds of 600 Daltons or less. Therefore, "improved ligands" are ligands that have better pharmacological and/or pharmacokinetic properties compared to the reference compound, where "better" can be used by persons who are used for specific biological systems or therapeutic purposes. definition.

In the context of binding compounds and ligands, the term "derivative" or "derivative compound" refers to a compound with the following chemical structure, which contains the common core chemical structure as the original compound or the reference compound, but it borrows It differs by having at least one structural difference, for example by adding and/or removing and/or replacing one or more substituents, and/or substituting one or more atoms with different atoms. Unless explicitly stated to the contrary, the term "derivative" does not mean the use of the original compound as a starting material or as an intermediate to synthesize a derivative, although in some cases the derivative can be synthesized from the original compound.

Therefore, the term "original compound" refers to a reference compound that has structural features that can also be found in derivative compounds. Often, but not always, the chemical structure of the original compound is simpler than that of the derivative.

"Chemical structure" or "chemical substructure" means any definable atom or group of atoms that constitute an individually identifiable part of a molecule, such as a substituent moiety, optionally substituted core and the like. The chemical substructure of the ligand can generally have the effect of binding the ligand to the target molecule, or can affect the three-dimensional shape, electrostatic charge, and/or configuration characteristics of the ligand.

Regarding the interaction between the target and the potential binding compound, the term "binding" means that the potential binding compound is bound to the target to a statistically significant degree compared to the general binding to a protein (ie, non-specific binding). Therefore, the term "binding compound" refers to a compound that has a statistically significant association with the target molecule. The binding compound preferably has a dissociation constant (K_D) Interact with a specific target. The binding compound can be combined as described herein with "low affinity", "very low affinity", "very low affinity", "medium affinity", "moderately high affinity" or "high affinity".

In the context of a compound that binds to a target, the term "greater affinity" means that the compound binds more tightly than the reference compound or the same compound under reference conditions (ie, having a lower dissociation constant). In certain embodiments, the greater affinity is at least 2, 3, 4, 5, 8, 10, 50, 100, 200, 400, 500, 1000, or 10,000 times higher than the affinity.

Also, in the context of a compound that binds to a biomolecular target, the term "greater specificity" means that the compound is more closely related to the specific target than another biomolecule or multiple biomolecules that can exist under the relevant binding conditions. Binding, where binding to these other biomolecules produces different biological activities than binding to a specific target. The specificity is usually related to a limited set of other biomolecules, such as in the case of c-kit, other tyrosine kinases, or other types of enzymes. In certain embodiments, the greater specificity is at least 2, 3, 4, 5, 8, 10, 50, 100, 200, 400, 500, or 1000 times higher than the specificity.

As used with regard to the binding of a compound to a target, the term "interaction" means that the distance from any atom of the binding compound to the specific amino acid residue will be 5.0 angstroms or less. In certain embodiments, the distance from the compound to the specific amino acid residue is 4.5 angstroms or less, 4.0 angstroms or less, or 3.5 angstroms or less. For example, co-crystallography can be used to determine these distances, or use computer fitting of compounds in the active site for evaluation.

As used herein, with regard to binding compounds or ligands, the terms "specific for c-kit kinase", "specific for c-kit" and terms with similar meanings mean that they are compared with other kinases that can occur in a specific organism , The specific compound binds to c-kit to a statistically greater degree. In addition, when expressing a biological activity different from binding, the term "specific to c-kit" means that compared with other tyrosine kinases, a specific compound has a greater biological effect associated with binding c-kit, such as kinase activity inhibition . The specificity preferably also relates to other biomolecules (not limited to tyrosine kinase) that can appear in the organism.

"Binding site" means the region of the target molecule to which a ligand can bind non-covalently. The binding site exhibits a specific shape and often contains multiple binding pockets that appear in the binding site. Certain shapes are often retained in molecular species (such as molecular families). The binding site in one class may also contain conserved structures, such as the chemical moiety, the presence of a binding pocket, and/or the electrostatic charge at the binding site or a part of the binding site, all of which can affect the shape of the binding site .

"Binding pocket" means a specific volume within a binding site. The binding pocket can often be a specific shape, gap or cavity in the binding site. The binding pocket may contain specific chemical groups or structures that are important in the non-covalent bonding of another molecule, such as groups that facilitate ionic bonding, hydrogen bonding, or intermolecular Van der Waals interactions.

With regard to a binding compound that binds to a target molecule, "oriented" means that the binding compound (which can be defined by referring to at least some of its constituent atoms) and the binding pocket and/or the space at least part of the target molecule defining the atoms of the binding pocket relation.

In the context of the target molecule in the present invention, the term "crystal" refers to a regular set of target molecules of the type suitable for X-ray crystallography. That is, when irradiated with an X-ray beam, the collection produces an X-ray diffraction pattern. Therefore, the crystal is distinguished from coalescence or other complexes of target molecules that cannot cause a diffraction pattern.

The phrase "change binding affinity or binding specificity" means to change the binding constant of the first compound to another compound, or to change the binding constant of the first compound to the second compound, respectively, compared with the degree of binding of the first compound to the third compound. The degree of integration. For example, if the relative degree of binding to a specific protein increases when compared to the binding of the compound to an unrelated protein, the binding specificity of the compound to that specific protein increases.

As used herein, with regard to test compounds, binding compounds, and modulators (ligands), the term "synthesis" and similar terms mean chemical synthesis from one or more precursor materials.

"Verification" means the creation of experimental conditions and the collection of specific results related to the experimental conditions. For example, enzymes can be tested based on their ability to act on a detectable substrate. Compounds or ligands can be tested based on their ability to bind to one or more specific target molecules.

The "group" of a compound means a collection of compounds. These compounds may or may not be structurally related.

In another aspect, providing a compound of formula I (such as a compound developed by the method described herein) that is active on c-kit also provides a method for regulating the activity of c-kit by contacting c-kit with a compound of formula I method. Preferably, the compound is provided to an extent sufficient to regulate the activity of c-kit by at least 10%, more preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90%. In many embodiments, the concentration of the compound will be about 1 μM, 100 μM, or 1 mM or in the range of 1-100 nM, 100-500 nM, 500-1000 nM, 1-100 μM, 100-500 μM, or 500-1000 μM. In certain embodiments, the contacting is performed outside the body.

As used herein, the term "modulating" or "modulate" refers to the effect of modifying biological activity (especially the biological activity associated with a specific biological molecule such as c-kit). For example, an agonist or antagonist of a specific biomolecule regulates the activity of the biomolecule (such as an enzyme).

In the context of using, testing, or screening for compounds that act or may act as modulators, the term "contact" means to bring the compound(s) sufficiently close to a specific molecule, complex, cell, tissue, organism, or other specific material, so that Potential binding interactions and/or chemical reactions can occur between the compound and other specific materials.

The bonding component may include, for example, a bonding agent (including a non-marking bonding agent) for bonding to a solid phase or another molecule or other part. The bond can be formed by synthesizing a compound or derivative on a linking agent that is bonded to a solid phase medium, for example, in the combined synthesis of multiple compounds. Likewise, binding to a solid phase medium can provide an affinity medium (for example, for affinity chromatography).

As used herein, with regard to an amino acid or nucleic acid sequence, the term "isolated" means to separate the sequence from at least a portion of the amino acid and/or nucleic acid sequence with which it is normally associated.

With regard to amino acid or nucleic acid sequences, the term "purified" means that the test molecule constitutes a significantly larger proportion of the biomolecules in the composition compared to the proportions observed in previous compositions (eg, in cell culture). The larger ratio can be 2 times, 5 times, 10 times, or more than 10 times relative to the ratios found in the previous composition.

From the following detailed description and the scope of the patent application, additional aspects and embodiments will be apparent.

<b>I. Summary</b>

The present invention provides a compound of formula I, which is a c-kit inhibitor and modulates the activity of c-kit. Table 1 shows exemplary compounds of formula I with anti-c-kit activity and their IC against c-kit₅₀The activity level is less than 1μM.

<b>Table 1</b>Provide the structure and name of a group of exemplary compounds of formula I that have activity on c-kit.

<tables><img file="TW200616632A_D0003.tif" /></tables>

<b>Exemplary diseases related to c-kit.</b>

The compounds described herein are suitable for the treatment of c-kit-related conditions, such as diseases related to improperly regulated kinase signal transduction, especially including cell proliferative conditions, fibrotic conditions, and metabolic conditions. As described in more detail below and Lipson et al., US20040002534 (U.S. Application No. 10/600,868, filed on June 23, 2003) (the entirety of which is incorporated herein by reference), it can be treated by the present invention The cell proliferative conditions include cancer and mast cell proliferative conditions.

As described below, the existence of c-kit has been linked to many different types of cancer. In addition, the link between c-kit abnormality and disease is not limited to cancer. For example, as also described in more detail below, c-kit has been associated with inflammatory diseases such as mastocytosis, asthma, multiple sclerosis, inflammatory bowel syndrome, and allergic rhinitis.

<b>Exemplary malignant diseases related to c-kit</b>Abnormal expression and/or activation of c-kit has been implied in various cancers. For example, evidence that c-kit contributes to tumor pathology includes its association with leukemia and mast cell tumors, small cell lung cancer, testicular cancer, and certain cancers of the gastrointestinal and central nervous system. In addition, it has been suggested that c-kit is involved in the carcinogenesis of female reproductive tract (Inoue et al., 1994,<i>Cancer Res</i>.54(11): 3049-3053), neuroectodermal-derived sarcoma (Ricotti et al., 1998,<i>Blood</i>91: 2397-2405) and the formation of Schwann cell tumors associated with multiple neurofibromas (Ryan et al., 1994,<i>J.Neuro.Res.</i>37:415-432) plays a certain role. We have found that mast cells are involved in altering the tumor microenvironment and enhancing tumor growth (Yang et al., 2003,<i>J Clin Invest.</i>112: 1851-1861; Viskochil, 2003,<i>J Clin Invest.</i>112:1791-1793). Therefore, c-kit is a useful target in the treatment of multiple neurofibromas and malignant tumors.

Small cell lung cancer: c-kit receptors have been found to be abnormally expressed in many cases of small cell lung cancer (SCLC) cells (Hibi et al., 1991,<i>Oncogene</i>6:2291-2296). Therefore, as an example, inhibition of c-kit may be beneficial in the treatment of SCLC, for example to improve the long-term survival of SCLC patients.

Leukemia: SCF bound to c-kit protects hematopoietic stem and progenitor cells from apoptosis (Lee et al., 1997,<i>J. Immunol.</i>159: 3211-3219), thereby contributing to community formation and blood cell production. In acute myeloid leukemia (AML) and in some cases of acute lymphoblastic leukemia (ALL), c-kit is often observed (for review, see Sperling et al., 1997,<i>Haemat</i>82: 617-621; Escribano et al., 1998,<i>Leuk.Lymph.</i>30: 459-466). Although c-kit is present in most AML cells, its performance does not seem to be a precursor to disease progression (Sperling et al., 1997,<i>Haemat</i>82: 617-621). However, SCF protects AML cells from apoptosis induced by chemotherapeutics (Hassan et al., 1996,<i>Acta.Hem.</i>95:257-262). Inhibition of c-kit by the present invention can enhance the efficacy of these drugs and can induce apoptosis of AML cells.

We have found that SCF combined with other cytokines can significantly enhance patients with myelodysplastic syndrome (Sawada et al., 1996,<i>Blood</i>88:319-327) or chronic myelogenous leukemia (CML) (Sawai et al., 1996,<i>Exp.Hem.</i>2: 116-122) the cell line growth of the patient. CML is characterized by the expansion of Philadelphia chromosome-positive cells in the bone marrow (Verfaillie et al., 1998,<i>Leuk.</i>12:136-138), which seems to be mainly caused by the inhibition of apoptotic death (Jones, 1997,<i>Curr.Opin.Onc.</i>9: 3-7). P210, a product of the Philadelphia chromosome, has been reported^B^C^R^－^A^B^LRegulate the inhibitory effect of apoptosis (Bedi et al., 1995,<i>Blood</i>86: 1148-1158) because of p210^B^C^R^－^A^B^LAnd c-kit both inhibit apoptosis and it has been shown that p62^d^o^kAs the substrate (Carpino et al., 1997,<i>Cell</i>88:197-204), so the pure lineage regulated by kinases can be expanded through the common signal transmission pathway. However, c-kit and p210 have also been reported^B^C^R^－^A^B^LDirect interaction (Hallek et al., 1996,<i>Brit.J Haem.</i>94:5-16), which shows that c-kit has a more causal role in CML pathology. Therefore, inhibition of c-kit will be useful in treating the above-mentioned conditions.

Gastrointestinal cancer: normal colorectal mucosa does not show c-kit (Bellone et al., 1997,<i>J. Cell Physiol.</i>172:1-11). However, c-kit is frequently expressed in colorectal cancer (Bellone et al., 1997,<i>J. Cell Physiol.</i>172:1-11), and the autocrine loop of SCF and c-kit has been observed in several colon cancer cell lines (Toyota et al., 1993,<i>Turn Biol</i>14: 295-302; Lahm et al., 1995,<i>Cell Growth &amp;Differ</i>6:1111-1118; Bellone et al., 1997,<i>J. Cell Physiol.</i>172:1-11). In addition, by using neutralizing antibodies to destroy the autocrine loop (Lahm et al., 1995,<i>Cell Growth &amp; Differ.</i>6:1111-1118) and down-regulation of c-kit and/or SCF can significantly inhibit cell proliferation (Lahm et al., 1995,<i>Cell Growth &amp; Differ</i>6:1111-1118; Bellone et al., 1997,<i>J. Cell Physiol.</i>172：1－11)。

The SCF/c-kit autocrine loop has been observed in gastric cancer cell lines (Turner et al., 1992,<i>Blood</i>80: 374-381; Hassan et al., 1998,<i>Digest.Dis.Science</i>43: 8-14), and constitutive c-kit activation also seems to be important for gastrointestinal stromal tumors (GIST). GIST is the most common mesenchymal tumor of the digestive system. More than 90% of GISTs express c-kit, which is consistent with the assumed origin of these tumor cells from Cajal interstitial cells (ICC) (Hirota et al., 1998,<i>Science</i>279: 577-580). We believe that ICC regulates gastrointestinal contraction, and patients lacking c-kit in ICC exhibit a form of chronic spontaneous intestinal pseudo-obstruction myopathy (Isozaki et al., 1997,<i>Amer.J.of Gast.</i>9332-334). It has been observed that c-kit expressed in GIST of several different patients has mutations in the intracellular membrane domain, leading to the constitutive activation of this RPTK (Hirota et al., 1998,<i>Science</i>279: 577-580). Therefore, inhibiting c-kit is an effective way to treat these cancers.

Testicular cancer: Male germ cell tumors have been classified histologically into seminoma (which retains germ cell characteristics) and non-seminoma (which shows characteristics of embryonic differentiation). We believe that both seminoma and non-seminoma start from the pre-spreading stage designated carcinoma in situ (CIS) (Murty et al., 1998,<i>Sem.Oncol.</i>25: 133-144). Both c-kit and SCF have been reported to be necessary for normal gonad development during embryogenesis (Loveland et al., 1997,<i>J. Endocrinol</i>153: 337-344). The loss of receptors or ligands can lead to animals lacking germ cells. In the testes after birth, c-kit has been found to express in Leydig cells and spermatogonia, while SCF is expressed in Stellite cells (Loveland et al., 1997,<i>J. Endocrinol</i>153: 337-344). In transgenic mice expressing the human papillomavirus 16 (HPV16) E6 and E7 oncogenes, testicular tumors developed from Leddy cells at a high frequency (Kondoh et al., 1991,<i>J. Virol.</i>65: 3335-3339; Kondoh et al., 1994,<i>J. Urol.</i>152:2151-2154). These tumors exhibit both c-kit and SCF, and the autocrine ring can contribute to tumorigenesis related to the loss of functional p53 cells (Kondoh et al., 1995,<i>Oncogene</i>10:341-347) and eye cancer gene products related to E6 and E7 (Dyson et al., 1989,<i>Science</i>243: 934-937; Werness et al., 1990,<i>Science</i>248: 76-79; Scheffner et al., 1990,<i>Cell</i>63: 1129-1136). SCF (Kondoh et al., 1995,<i>Oncogene</i>10:341-347) or c-kit (Li et al., 1996, Canc. Res. 56:4343-4346) defective signal transmission mutants inhibit the formation of testicular tumors in mice expressing HPV16 E6 and E7. The activation of c-kit kinase is critical to the tumorigenesis of these animals. Therefore, the regulation of the c-kit kinase pathway by the present invention can prevent or treat these conditions.

The expression effect of c-kit in germ cell tumors has been shown to express receptors in most carcinomas in situ and seminoma, but c-kit is only expressed in a few non-seminomas (Strohmeyer et al., 1991,<i>Canc.Res.</i>51: 1811-1816; Rajpert-de Meyts et al., 1994,<i>Int.J.Androl.</i>17:85-92; Izquierdo et al., 1995,<i>J. Pathol.</i>177:253-258; Strohmeyer et al., 1995,<i>J. Urol.</i>153: 511-515; Bokenmeyer et al., 1996,<i>J. Cancer Res. Clin. Oncol.</i>122: 301-306; Sandlow et al., 1996,<i>J. Androl.</i>17: 403-408). Therefore, inhibition of c-kit provides a way to treat these conditions.

CNS cancer: SCF and c-kit are expressed in the entire CNS of developing rodents, and the expression pattern indicates their role in the growth, migration and differentiation of neuroectoderm cells. The performance of both receptors and ligands in the adult brain has been reported (Hamel et al., 1997,<i>J. Neuro</i>－<i>Onc.</i>35：327－333)_The performance of c-kit has also been observed in normal human brain tissue (Tada et al., 1994,<i>J. Neuro</i>80: 1063-1073). The glioblastoma and astrocytoma lines that define most intracranial tumors result from tumor transformation of astrocytes (Levin et al., 1997, Principles &amp; Practice of Oncology: 2022-2082). The performance of c-kit has been observed in glioblastoma cell lines and tissues (Berdel et al., 1992,<i>Canc.Res.</i>52: 3498-3502; Tada et al., 1994,<i>J. Neuro</i>80: 1063-1073; Stanulla et al., 1995,<i>Act Neuropath</i>89：158－165)。

Cohen et al., 1994,<i>Blood</i>84: 3465-3472 reported that all 14 tested neuroblastoma cell lines contained the c-kit/SCF autocrine loop, and both receptors and ligands were observed in 45% of the tested tumor samples Performance. In both cell lines, anti-c-kit antibodies inhibited cell proliferation, which indicates that the SCF/c-kit autocrine loop contributes to growth (will Cohen et al., 1994, Blood 84: 3465-3472). Therefore, c-kit inhibitors can be used to treat these cancers.

<b>Exemplary mast cell disease involving c-kit</b>The excessive activation of c-kit is also related to diseases caused by excessive mast cells. Mastocytosis is a term used to describe a heterogeneous group of conditions characterized by excessive mast cell proliferation (Metcalfe, 1991,<i>J.Invest.Derm</i>93: 2S-4S; Golkar et al., 1997,<i>Lancet</i>349: 1379-1385). The high c-kit expression on mast cells of patients with aggressive mastocytosis was reported (Nagata et al., 1998,<i>Leukemia</i>12：175－181)。

In addition, mast cells and eosinophils represent key cells involved in allergy, inflammation, and asthma (Thomas et al., 1996,<i>Gen.Pharmacol</i>27:593-597; Metcalfe et al., 1997,<i>Physiol Rev</i>77: 1033-1079; Naclerio et al., 1997,<i>JAMA</i>278:1842-1848; Costa et al., 1997,<i>JAMA</i>278: 1815-1822). SCF and thus c-kit directly and indirectly regulate the activation of both mast cells and eosinophils, thereby affecting the primary cells involved in allergies and asthma through a variety of mechanisms. Due to the mutual regulation of the functions of mast cells and eosinophils and the role of SCF in this regulation, the inhibitory effect of c-kit can be used to treat chronic rhinitis, inflammation and asthma related to allergies.

Mastocytosis: It has been reported that the stimulating effect of SCF (also known as mast cell growth factor) on c-kit is necessary for the growth and development of mast cells (Hamel et al., 1997,<i>J. Neuro</i>－<i>Onc.</i>35: 327-333; Kitamura et al., 1995,<i>Int.Arch.Aller.Immunol.</i>107: 54-56). Mice with the c-kit mutation that impairs their signal transmission activity have been significantly shown to have fewer mast cells in their skin (Tsujimura, 1996,<i>Pathol Int</i>46: 933-938). Excessive activation of c-kit may be related to diseases caused by too many mast cells.

Mastocytosis is limited to the skin of most patients, but it can involve other organs in 15-20% of patients (Valent, 1996,<i>Wein</i>/<i>Klin Wochenschr</i>108: 385-397; Golkar et al., 1997,<i>Lancet</i>349: 1379-1385). Even in patients with systemic mastocytosis, the disease ranges from a relatively benign prognosis to aggressive mastocytosis and mast cell leukemia. (Valent, 1996,<i>Wein/Klin Wochenschr</i>108: 385-397; Golkar et al., 1997,<i>Lancet</i>349: 1379-1385). Malignant mast cells in canine mast cell tumors (London et al., 1996,<i>J.Compar.Pathol.</i>115:399-414) and mast cells in patients with aggressive systemic mastocytosis (Baghestanian et al., 1996,<i>Leuk.</i>: 116-122; Castells et al., 1996,<i>J.Aller.Clin.Immunol.</i>98: 831-840) c-kit has been observed.

It has been shown that SCF behaves as a cell membrane-bound protein on stromal cells, and its performance can be induced by fibrogenic growth factors such as PDGF (ie, platelet-derived growth factor). And it has been shown that it appears as a cell membrane-bound protein on keratinocytes in normal skin. However, in the skin of patients with mastocytosis, an increase in the amount of soluble SCF has been observed (Longley et al., 1993,<i>New Engl.J.Med.</i>328：1302－1307)。

It has been reported that mast cell chymosin cleaves SCF associated with cell membranes into a soluble and biologically active form. This process regulated by mast cells can generate a feedback loop to enhance the proliferation and function of mast cells (Longley et al., 1997,<i>Proc.Natl.Acad.Sci.</i>94:9017-9021), and it is important for the etiology of mastocytosis. Transgenic mice that overexpress SCF, a form of SCF that cannot be released from keratinocytes by proteolysis, do not develop mastocytosis, and similar animals that exhibit normal SCF in keratinocytes exhibit similarities to human skin mast cells The phenotype of hyperplasia (Kunisada et al., 1998,<i>J.Exp.Med.</i>187: 1565-1573). The formation of a large amount of soluble SCF can contribute to the pathology associated with mastocytosis in some patients, and the present invention can treat or prevent these conditions by regulating the interaction between SCF and c-kit. Several different c-kit mutations that lead to constitutive kinase activity have been found in human and rodent mast cell tumor cell lines (Furitsu et al., 1993,<i>J.Clin.Invest.</i>92:1736-1744; Tsujimura et al., 1994,<i>Blood</i>9:2619-2626; Tsujimura et al., 1995, Int.Arch.Aller.Immunol 106:377-385; Tsujimura, 1996,<i>Pathol Int</i>46: 933-938). In addition, peripheral monocytes have been isolated from patients with mastocytosis and related hematological conditions (Nagata et al., 1998,<i>Mastocytosis Leuk</i>12:175-181) Neutralize mast cells in patients with pigmented urticaria and aggressive mastocytosis (Longley et al., 1996,<i>Nat.Gen.</i>12:312-314) an activating mutation of the c-kit gene was observed. Therefore, it is proved that the inhibition of c-kit has an excellent therapeutic effect in the treatment of these diseases.

In some patients, activating mutations of c-kit can be responsible for the pathogenesis of the disease, and can treat these patients or prevent their diseases by regulating the interaction between SCF and c-kit. It has been shown that the SCF activation effect of c-kit prevents mast cell apoptosis, which is crucial for maintaining the homeostasis of skin mast cells (Iemura et al.,<i>Amer.J.Pathol</i>1994,<b>144</b>: 321-328; Yee et al.,<b>J.Exp.Med.</b>1994,<b>179</b>: 1777-1787; Mekori et al.,<i>J. Immunol</i>1994,<b>153</b>: 2194-2203; Mekori et al.,<i>Int.Arch.Allergy Immunol.</i>1995,<b>107</b>: 137-138). Inhibition of mast cell apoptosis can lead to the accumulation of mast cells associated with mastocytosis. Therefore, observing the activation of c-kit caused by the overexpression of the receptor, the over-formation of soluble SCF, or the mutation of the c-kit gene that constitutionally activates its kinase can lead to the following conclusions: inhibition of c-kit kinase activity reduces hypertrophy The number of cells and provide benefits for patients with mastocytosis.

For cells with activating c-kit mutations, c-kit inhibitors have been found to inhibit or even kill these cells (Ma et al., 2000,<i>J Invest Dermatol.</i>114:392-394), especially for mutations in regulatory regions (Ma et al., 2002,<i>Blood</i>99:1741-1744). Ma et al., 2002 also showed that for mutations in the catalytic region, inhibitors STI571 (Gleevec) and SU9529 do not inhibit cells, making additional types of c-kit inhibitors applicable. Therefore, c-kit inhibitors can be used to combat wild-type c-kit and c-kit with mutations (such as activating mutations) in the regulatory region and/or in the catalytic region.

Asthma and allergies: mast cells and eosinophils represent key cells in indications such as parasitic infection, allergy, inflammation and asthma (Thomas et al., Gen. Pharmacol 1996,<b>27</b>: 593-597; Metcalfe et al., Physiol Rev 1997,<b>77</b>: 1033-1079; Holgate, 1997, CIBA Found. Symp.; Naclerio et al., JAMA 1997,<b>278</b>:1842-1848; Costa et al., JAMA 1997,<b>778</b>: 1815-1822). It has been shown that SCF is necessary for the development, survival and growth of mast cells (Kitamura et al., 1995,<i>Int.Arch.Aller.Immunol.</i>107: 54-56; Metcalfe et al., 1997,<i>Physiol Rev</i>77: 1033-1079). In addition, SCF cooperates with eosinophil specific regulatory factor (IL-5) to increase the development of eosinophil progenitor cells (Metcalf et al., 1998, Proc. Natl. Acad. Sci., USA 95: 6408-6412). It has also been reported that SCF induces mast cell secretion and promotes the survival of eosinophils (Kay et al., 1997, Int.Arch.Aller.Immunol.113:196-199) (Okayama et al., 1997, Int.Arch.Aller.Immunol). 114: 75-77; Okayama et al., 1998, Eur. J. Immunol. 28: 708-715), which may contribute to chronic eosinophilic regulation inflammation (Okayama et al., 1997, Int. Arch. Aller Immunol. 114: 75-77; Okayama et al., 1998, Eur. J. Immunol. 28: 708-715). In this regard, SCF directly and indirectly regulates the activation of both mast cells and eosinophils.

SCF induces the following processes: the release of mediators from mast cells and the initiation of these cells for degranulation induced by IgE (Columbo et al., 1992, J. Immunol 149: 599-602) and make it resistant to eosinophils. Red blood cell particles become sensitive to the reactivity of the main basic protein (Furuta et al., 1998, Blood 92: 1055-1061). Among the factors released by activated mast cells are IL-5, GM-CSF and TNF-α, which affect the secretion of eosinophil protein (Okayama et al., 1997, Int.Arch.Aller.Immunol.114:75-77; Okayama et al., 1998, Eur. J. Immunol. 28:708-715). In addition to inducing the release of histamine from mast cells (Luckacs et al., 1996, J. Immunol. 156: 3945-3951; Hogaboam et al., 1998, J. Immunol. 160: 6166-6171), SCF also promotes eosinophilia. Mast cell production of eotaxin (Hogaboam et al., 1998, J. Immunol. 160: 6166-6171) and eosinophil infiltration (Luckacs et al., 1996, J. Immunol. 156: 3945-3951) .

SCF also directly affects mast cells (Dastych et al., 1994, J. Immunol. 152: 213-219; Kinashi et al., 1994, Blood 83: 1033-1038) and eosinophils (Yuan et al., 1997, J. Exp. Med. 186: 313-323) the adhesion of the two, which in turn regulates tissue infiltration. Therefore, SCF can affect the native cells involved in allergies and asthma through a variety of mechanisms. Currently, corticosteroids are the most effective in treating chronic rhinitis and inflammation related to allergies (Naclerio et al., 1997,<i>JAMA</i>278:1842-1848; Meltzer, 1997,<i>Aller.</i>52: 33-40). These agents act through a variety of mechanisms, including reducing the circulation and infiltration of mast cells and eosinophils, and reducing the survival rate of eosinophils that are related to the suppression of cytokine production (Meltzer, 1997,<i>Aller.</i>52: 33-40). It has also been reported that steroids inhibit the expression of SCF by fibroblasts and resident connective tissue cells, which leads to a decrease in the survival rate of mast cells (Finotto et al., 1997,<i>J.Clin.Invest.</i>99 1721-1728). Because of the mutual regulation of the functions of mast cells and eosinophils and the role of SCF in this regulation, the inhibitory effect of c-kit provides a way to treat chronic rhinitis, inflammation and asthma related to allergies.

Inflammatory arthritis (e.g. rheumatoid arthritis): due to the link between mast cells and arthritis progression (Lee et al., 2002,<i>Science</i>297:1689-1692), c-kit provides useful targets for the prevention, delay and/or treatment of inflammatory arthritis (such as rheumatoid arthritis).

Multiple sclerosis: As demonstrated in mouse models of experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS), it has been shown that mast cells play a broad role in autoimmune diseases. It shows that mast cells are necessary for the full manifestation of the disease. Secor et al., 2000,<i>J Exp</i>Med 191: 813-821. Therefore, c-kit also provides useful targets for the prevention, delay and/or treatment of multiple sclerosis.

Therefore, modulators of c-kit function can be used to combat diseases such as those shown above.

<b>II. Combination test</b>

The method of the present invention includes an assay capable of detecting the binding of a compound to a target molecule. The combination is at a statistically significant degree, and it has a credibility of preferably at least 90%, more preferably at least 95, 97, 98, 99% or greater. Combine (ie, distinguish from the background). It is preferable to use a control to distinguish target binding from non-specific binding. The assay of the present invention may also include assay compounds for binding to target molecules with low affinity. We have known many kinds of tests that are used in combination of different target types and these tests can be used in the practice of the present invention. Due to the broad nature of its binding, compounds with a wide range of actions across protein families cannot have high affinity for individual targets. Therefore, the assay described herein allows the identification of compounds that bind with low, very low, and very low affinity.

Binding with "low affinity" means that under standard conditions with a dissociation constant (K_D) Binding to the target molecule. Binding with "very low affinity" means a K over about 100μM under standard conditions_DCombine. Binding with "very low affinity" means that under standard conditions with a K of more than about 1 mM_DCombine. Binding with "medium affinity" means a K of about 200 nM to about 1 μM under standard conditions_DBinding: Binding with "moderately high affinity" means a K of about 1 nM to about 200 nM_DCombine. Binding with "high affinity" means under standard conditions with a K of less than about 1 nM_DCombine. For example, relative to the situation in which higher affinity binding occurs, low affinity binding can also occur because of the lower conformity to the binding site of the target molecule, or because the number of non-covalent bonds is small, or it may cause coordination. The covalent bond existing between the position and the binding site of the target molecule is weak. The standard conditions for binding were pH 7.2 at 37°C for one hour. For example, 100 μl/well can be maintained in HEPES 50 mM buffer, NaCl 15 mM, ATP 2 μM, and bovine serum albumin 1 μg/well at 37° C. at pH 7.2 for one hour.

The binding compound can also be characterized by its effect on the activity of the target molecule. Therefore, under standard conditions, the inhibitory concentration (IC₅₀) Or excitation concentration (EC₅₀) Is greater than 1μM. "Very low activity" means IC under standard conditions₅₀Or EC₅₀Above 100μM. "Very low activity" means IC under standard conditions₅₀Or EC₅₀Above 1 mM. "Moderately active" means IC under standard conditions₅₀Or EC₅₀It is 200 nM to 1 μM. "Moderately high activity" means IC₅₀Or EC₅₀From 1 nM to 200 nM. "High activity" means IC under standard conditions₅₀Or EC₅₀Less than 1 nM. IC₅₀(Or EC₅₀) Is defined as the concentration of the compound at which the activity of the target molecule (such as an enzyme or other protein) to be measured loses (or increases) 50% of the activity relative to the activity in the absence of the compound. The activity can be measured by methods known to those skilled in the art, for example, by measuring any detectable product or signal produced by the enzymatic reaction or other activity of the protein to be measured.

The "background signal" for the binding assay means the signal recorded under the standard conditions for the specific assay for the absence of a test compound or ligand that binds to the target molecule. Those who are familiar with this technique should be aware that there are acceptable methods and they are widely used to measure background signals.

"Standard deviation" means the square root of the variance. Variance is how to spread out the distribution. It is calculated as the average square deviation of each number from its average. For example, for numbers 1, 2, and 3, the mean is 2 and the variance is:<maths><img file="TW200616632A_D0004.tif" /></maths>

The related protein can be tested against a set of compounds or groups. These assays may preferably be enzymatic or combined assays. In some embodiments, it may be necessary to enhance the solubility of the compound to be screened and then analyze all compounds that exhibit activity in the assay, including compounds that bind with low affinity or generate a signal that is about three times greater than the standard deviation of the background signal. The assays can be any suitable assays, such as binding assays that measure the binding affinity between two binding partners. The different types of screening tests that can be used in the practice of the present invention are known in the art, such as the tests described in U.S. Patent Nos. 5,763,198, 5,747,276, 5,877,007, 6,243,980, 6,294,330, and 6,294,330, the full text of each Incorporated into this article by reference, including all diagrams and drawings.

In various embodiments of the assays, at least one compound, at least about 5%, at least about 10%, at least about 15%, at least about 20%, or at least about 25% of the compounds can bind with low affinity. Generally speaking, up to about 20% of the compounds in the screening assay can display activity, and then high-yield co-crystallography and computational analysis can be used to directly analyze these compounds to group these compounds into common structural characteristics (Such as structural core and/or shape and polarity features) categories and identify common chemical structures between compounds that exhibit activity.

<b>Measure the enzymatic and binding reaction during the screening test</b>Techniques for measuring, for example, the progress of enzymatic and binding reactions in multi-sleeve carriers are known in the art and include (but are not limited to) the following techniques.

Spectrophotometry and spectrofluorometric verification are well known in this technology. Examples of such tests include the use of chromaticity tests to detect peroxides, such as Gofdon, AJ and Ford, RA, (1972)<b>The Chemist's Companion: A Handbook Of Practical Data, Techniques, And References</b>, John Wiley and Sons, NY, page 437.

Fluorescence spectrometry can be used to monitor reaction product production. Fluorescence methodology is generally more sensitive than absorption methodology. Those who are familiar with this technique are familiar with the use of fluorescent probes. For comments, see Bashford et al. (1987)<b>Spectrophotometry and Spectrofluorometry: A Practical Approach</b>, Pages 91-114, IRL Press Ltd.; and Bell, (1981)<b>Spectroscopy In Biochemistry</b>, Volume I, pages 155-194, CRC Press.

In the spectrofluorometric method, the enzyme is exposed to a substrate that changes its intrinsic fluorescence when treated with the target enzyme. The matrix is usually non-fluorescent and is converted to a fluorophore through one or more reactions. As a non-limiting example, Amplex can be used<img file="TW200616632A_D0005.tif" />Red reagent (Molecular Probes, Eugene, OR) detects SMase activity. In order to use Amplex<img file="TW200616632A_D0006.tif" />Red measures the activity of sphingomyelinase, and the following reactions occur. First, SMase hydrolyzes sphingomyelin to produce ceramide and phosphocholine. Second, alkaline phosphatase hydrolyzes phosphorylcholine to generate choline. Third, choline oxidase is used to oxidize choline to betaine. Finally, make H in the presence of horseradish peroxidase₂O₂With Amplex<img file="TW200616632A_D0007.tif" />Red reacts to generate the fluorescent product Resorufin, and the signal from it is detected by spectrofluorometry.

Fluorescence polarization method (FP) is based on the reduction of the molecular rotation speed of the fluorophore that occurs when bound to larger molecules (such as receptor proteins), allowing the bound ligands to emit polarized fluorescence . By measuring the vertical and horizontal components emitted by the fluorophore and then excited by plane polarized light, the FP is mainly determined empirically. When the rotation of the molecules of the fluorophore is reduced, the polarized emission increases. When the fluorophore binds to a larger molecule (ie, the acceptor) to slow down the rotation of the fluorophore molecule, it generates a larger polarization signal. The magnitude of the polarized signal is quantitatively related to the degree of binding of the fluorescent ligand. Therefore, the polarization of the "binding" signal depends on the maintenance of high-affinity binding.

FP is an equalization technology and the response is very fast, it takes several seconds to several minutes to reach equilibrium. These reagents are stable and can be prepared in large quantities, resulting in high reproducibility. Because of these characteristics, FP has been proven to be highly automated, and a single incubation with a single premixed tracer-receptor reagent is usually used to perform FP. For comments, see Owicki et al., (1997), Application of Fluorescence Polarization Assays in High-Throughput Screening,<i>Genetic Engineering News</i>,17：27。

Especially FP is needed because its reading is independent of emission intensity (Checovich, WJ et al., (1995)<i>Nature</i>375: 254-256; Dandliker, WB et al. (1981)<i>Methods in Enzymology</i>74: 3-28) and is therefore not sensitive to the presence of colored compounds that stop fluorescent emission. FP and FRET (see below) are well suited to identify compounds that block the interaction between sphingolipid receptors and their ligands. For example, see Parker et al. (2000) Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays, J Biomol Screen 5:77-88.

Fluorophores derived from sphingolipids that can be used in FP assays are commercially available. For example, Molecular Probes (Eugene, OR) currently sells sphingomyelin and a sphingomyelin fluorophore. These substances are N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-dicyclopentadiene and benzene-3-pentanyl) Sphingosine Phosphocholine (BODIPY<img file="TW200616632A_D0008.tif" />FL C5-sphingomyelin); N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-dicyclopentadiene benzene-3-ten Diacyl) sphingosine phosphorylcholine (BODIPY<img file="TW200616632A_D0009.tif" />FL C12-sphingomyelin); and N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-s-dicyclopentadiene benzene-3- Pentyl) sphingosine (BODIPY<img file="TW200616632A_D0010.tif" />FL C5-Ceramide). US Patent No. 4,150,949 (Immunoassay for Gentamicin) discloses luciferin-labeled gentamicin, including luciferin thiamine methyl gentamicin. Additional fluorophores can be prepared by methods well known to the skilled person.

Exemplary normal and polarized fluorescence readers include POLARION<img file="TW200616632A_D0011.tif" />Fluorescence polarization system (Tecan AG, Hombrechtikon, Switzerland). Can use general multi-well plate reader for other verification, such as VERSAMAX<img file="TW200616632A_D0012.tif" />Reader and SPECTRAMAX<img file="TW200616632A_D0013.tif" />Multiwell disk spectrophotometer (both purchased from Molecular Devices).

Fluorescence resonance energy transfer (FRET) is another useful test for detecting interactions and has been described. See, for example, Heim et al. (1996)<i>Curr.Biol.</i>6: 178-182; Mitra et al. (1996)<i>Gene</i>173: 13-17; and Selvin et al. (1995)<i>Meth.Enzymol.</i>246: 300-345. FRET detects the energy transfer between two fluorescent substances in close proximity, which have known excitation and emission wavelengths. As an example, the protein can be expressed as a fusion protein with green fluorescent protein (GFP). When two fluorescent proteins approach, such as when the protein specifically interacts with a target molecule, resonance energy can be transferred from one excited molecule to another. As a result, the emission spectrum of the sample is converted, which can be measured by a fluorometer such as an fMAX porous fluorometer (Molecular Devices, Sunnyvale Calif).

The scintillation proximity assay (SPA) is a particularly useful assay for detecting the interaction with the target molecule. SPA is widely used in the pharmaceutical industry and has been described (Hanselman et al., (1997)<i>J. Lipid Res.</i>38: 2365-2373; Kahl et al. (1996)<i>Anal.Biochem.</i>243:282-283; Undenfriend et al. (1987)<i>Anal.Biochem.</i>161: 494-500). See also U.S. Patent Nos. 4,626,513 and 4,568,649 and European Patent No. 0,154,734. A commercially available system uses FLASHPLATE<img file="TW200616632A_D0014.tif" />Scintillator coated discs (NEN Life Science Products, Boston, MA).

The target molecules can be bound to the scintillator disk in various well-known ways. Scintillator disks that have been derivatized to bind to fusion proteins, such as GST, His6, or Flag fusion proteins, are commercially available. When the target molecule is a protein complex or multimer, a protein or subunit can be attached to the disk first, and then other components of the complex are subsequently added under binding conditions, resulting in the formation of a binding complex.

In a typical SPA assay, the gene product in the expression cell has been labeled with a radioisotope and added to the wells and allowed to interact with the solid phase, which is the immobilized target molecule and scintillator in the well coating. The verification can be immediately measured or brought to equilibrium. Either way, when the radioisotope label is sufficiently close to the scintillator coating, it can be produced by such as TOPCOUNT NXT<img file="TW200616632A_D0015.tif" />The signal detected by the device of the mini-disk scintillation counter (Packard BioScience Co., Meriden Conn.). If the radioisotope-labeled performance product binds to the target molecule, the radioisotope label and the scintillator remain close enough to generate a detectable signal.

In contrast, labeled proteins that are not bound to the target molecule or that are bound only briefly will not stay close to the scintillator long enough to generate the above-mentioned background signal. Any time spent near the scintillator caused by random Brownian motion will not result in a significant amount of signal. Likewise, there may be residual unincorporated radioisotope labels used during the presentation step, but will not produce a significant signal because it will be in solution rather than interacting with the target molecule. Therefore, these non-binding interactions will result in a certain degree of background signal that can be arithmetic removed. If too much signal is obtained, salt or other modifiers can be directly added to the test plate until the desired specificity is obtained (Nichols et al., (1998)<i>Anal.Biochem.</i>257：112－119)。

<b>III. Kinase Activity Assay</b>

Many different kinase activity assays can be used to test activity modifiers and/or determine the specificity of modulators for a particular kinase or group of kinases. In addition to the tests described in the following examples, an ordinary person familiar with this technology should understand the other tests available and can modify the tests for specific applications. For example, many papers on kinases describe the assays that can be used.

C-kit purified by the procedure described in the examples can be used according to the following procedures to perform kinase activity assays that can be used for c-kit. Additional alternative assays can use binding assays. For example, by changing the donor and acceptor reagents or phosphorus-specific antibodies linked to streptavidin, you can use the fluorescence resonance energy transfer (FRET) format or use the AlphaScreen (amplified luminescence approximation uniformity test) format To format this type of test.

<b>IV. Organic Synthesis Technology</b>

In this technology, there are a variety of organic synthesis technologies that facilitate the construction of the compounds of the present invention. Many of these organic synthesis methods are described in detail in standard reference sources used by those familiar with the technology. An example of this kind of reference is March, 1994,<b>Advanced Organic Chemistry; Reactions,Mechanisms and Structure</b>, New York, McGraw Hill. Therefore, those who are familiar with organic chemical synthesis can easily use the technology suitable for the synthesis of potential modulators of kinase function.

<b>V. Administration</b>

These methods and compounds will generally be used in the treatment of human subjects. However, it can also be used to treat similar or identical diseases of other vertebrates, such as other primates, sports animals, and pets, such as horses, dogs, and cats.

The appropriate dosage form depends in part on the use or route of administration, such as oral, transdermal, transmucosal, inhalation or injection (parenteral). These dosage forms should allow the compound to reach the target cells. Other factors are well known in the art and include considerations such as toxicity and dosage form that delay the compound or composition from exerting its effects. Generally available at<i>Remington's Pharmaceutical Sciences</i>, 18th edition, Mack Publishing Co., Easton, PA, 1990 (by which it is incorporated herein by reference) techniques and formulations are found.

The compounds can be formulated as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are non-toxic salts in their dosage and concentration. By changing the physical characteristics of the compound without preventing the compound from exerting its physiological effects, the preparation of these salts can be convenient for pharmacological use. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate the administration of higher concentrations of drugs.

Pharmaceutically acceptable salts include acid addition salts, such as containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, Acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate and quinicate kind. It can be selected from hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, aminosulfonic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, The acids of cyclohexylaminosulfonic acid, fumaric acid and quinic acid are pharmaceutically acceptable salts.

When there are acidic functional groups such as carboxylic acid or phenol, pharmaceutically acceptable salts also include base addition salts, such as containing benzathine, chloroprocaine, choline, diethanolamine , Ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine and zinc salts. See for example<i>Remington's Pharmaceutical Sciences</i>, 19th edition, Mack Publishing Co., Easton, PA, Volume 2, Page 1457, 1995. These salts can be prepared using appropriate corresponding bases.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of the compound is dissolved in a suitable solvent (such as an aqueous solution containing a suitable acid or a water-alcohol solution) and then separated by evaporating the solution. In another example, the salt is prepared by reacting the free base with an acid in an organic solvent.

Pharmaceutically acceptable salts of different compounds can exist as complexes. Examples of the complex include 8-chlorotheophylline complex (similar to, for example, dimenhydrinate: diphenylhydrolamide 8-chlorotheophylline (1:1) complex; Dramamine) ) And various cyclodextrin inclusion complexes.

Carriers or excipients can be used to produce the composition. The carrier or excipient can be selected to facilitate administration of the compound. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (such as lactose, glucose or sucrose) or various types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution, and dextrose.

Compounds can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, transrectal, transdermal, or inhalation. Oral administration is better. For example, for oral administration, the compound can be formulated into conventional oral dosage forms such as capsules, lozenges, and liquid preparations such as syrups, elixirs, and concentrated drops.

Preparations for oral use are available, for example, by combining the active compound with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if necessary, to obtain lozenges or dragee cores. Suitable excipients are especially fillers, such as sugars, including lactose, sucrose, mannose or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, and agar Base cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose (CMC) and/or polyvinylpyrrolidone (PVP: polyvinypyrone). If necessary, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (such as sodium alginate).

The core of the dragee is provided with a suitable coating. For this purpose, a concentrated sugar solution can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyacrylic acid gel, polyethylene glycol (PEG) and/or titanium dioxide, lacquer solutions and suitable Organic solvent or solvent mixture. Dyestuffs or pigments may be added to the coatings of the tablets or dragees for identification or to characterize different combinations of active compound doses.

Oral preparations include cooperating insert capsules ("caps") made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Compatible insert capsules may contain active ingredients mixed with fillers (such as lactose), binders (such as starch) and/or lubricants (such as talc or magnesium stearate) and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol (PEG). In addition, stabilizers can be added.

Alternatively, injection (parenteral administration) may be used, for example intramuscularly, intravenously, intraperitoneally, and/or subcutaneously. For injection, the compound of the present invention can be formulated in a sterile liquid solution, preferably in a physiologically compatible buffer or solution, such as saline solution, Hank's solution or Ringer's solution (Ringer's solution). In addition, the compound can be formulated into a solid form and re-dissolved or suspended immediately before use. It can also be produced in freeze-dried form.

It can also be administered via mucosal, transdermal or inhalation. For transmucosal, transdermal or inhalation administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art, and for transmucosal administration, they include, for example, bile salts and fusidic acid derivatives. In addition, detergents can be used to promote penetration. For example, transmucosal administration can be via nasal spray or suppository (rectal or vaginal).

For inhalation, the compound of the present invention can be formulated as a dry powder or a suitable solution, suspension or aerosol. Powders and solutions can be formulated with suitable additives known in the art. For example, the powder may include a suitable powder base, such as lactose or starch, and the solution may contain propylene glycol, sterile water, ethanol, sodium chloride, and other additives such as acids, bases, and buffer salts. These solutions or suspensions can be administered by inhalation through sprays, pumps, nozzles or nebulizers and the like. The compound of the present invention can also be used in combination with other inhaled therapeutic agents, such as corticosteroids, such as fluticasone proprionate, beclomethasone dipropionate, triamcinolone acetonide, Budesonide and mometasone furoate furoate); beta agonists, such as albuterol, salmeterol and formoterol; anticholinergic agents, such as ipratropium bromide or tiotropium; vasodilation Agents, such as treprostinal and iloprost; enzymes, such as DNAase; therapeutic proteins; immunoglobulin antibodies; oligonucleotides, such as single or double-stranded DNA or RNA, siRNA; antibiotics, Such as tobramycin; muscarinic receptor antagonist; leukotriene antagonist; cytokine antagonist; protease inhibitor; cromolyn sodium; nedocril sodium; and cromolyn sodium.

It should be understood that combined use includes the delivery of the compound of the present invention and one or more other inhaled therapeutic agents in any formulation, which formulation includes a formulation in which the two compounds are chemically linked so that they can maintain their therapeutic activity during administration. . The combined use includes the administration of co-formulations or chemically combined compound formulations or the co-administration of compounds in separate formulations. The individual formulations can be co-administered by delivery from the same inhalation device, or can be co-administered from separate inhalation devices, where co-administration in this case means mutual administration in a short time. The co-formulation of the compound of the present invention and one or more additional inhaled therapeutic agents includes preparing the materials together so that they can be administered through an inhalation device that includes individual compounds combined in a formulation or modified to It is a compound that is chemically bound but still maintains its biological activity.

Use standard procedures to determine the amount of each compound to be administered, considering the following factors: such as compound IC₅₀, The biological half-life of the compound, the age, size and weight of the patient, and the symptoms related to the subject. Those who are familiar with this technology are familiar with the importance of these and other factors. Generally speaking, the dosage will be between about 0.01 and 50 mg/kg of the treated subject, preferably between 0.1 and 20 mg/kg of the treated subject. Multiple doses can be used.

<b>VI. Operation c-kit</b>

Since the full-length coding sequence and amino acid sequence of c-kit of various mammals including humans are already known, it is easy to perform the selection and construction of recombinant c-kit, the production and purification of recombinant protein, and the entry of c-kit Introduction in other organisms and other c-kit molecular biology operations.

The techniques for manipulating nucleic acids are fully disclosed in scientific and patent literature, such as sub-colonization, labeled probes (for example, random primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and similar techniques, for example, see Molecular Cloning edited by Sambrook: a Laboratory Manual (Second Edition), Volumes 1-3, Cold Spring Harbor Laboratory, (1989); Current Protocols in Molecular Biology, edited by Ausubel. John Wiley &amp; Sons, Inc., New York (1997); Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, edited by Tijssen. Elsevier, NY (1993).

When necessary, amplification methods (such as PCR, isothermal method, rolling circle method, etc.) can be used to amplify the nucleic acid sequence for further use, and these methods are well known to the skilled person. For example, see Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al., eds., Academic Press, San Diego, CA 1990, pp. 13-20; Wharam et al.,<i>Nucleic Acids Res.</i>June 1, 2001; 29(11): E54-E54; Hafner et al.,<i>Biotechniques</i>April 2001; 30(4): 852-6,858,860 passim; Zhong et al.,<i>Biotechniques</i>April 2001; 30(4): 852-6,858,860 passim.

Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified in any of a variety of general methods well known to those skilled in the art. These methods include, for example, analytical biochemical methods, such as NMR, spectrophotometry, X-ray photography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and high Diffusion chromatography; various immunological methods, such as fluid or gel precipitation reaction method, immunodiffusion method, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELIsA), immunofluorescence assay Method, southern analysis method, northern analysis method, ink spot analysis method, gel electrophoresis method (such as SDS-PAGE), nucleic acid or target or signal amplification method, isotope labeling method, scintillation counting method and affinity chromatography.

The nucleic acid acquisition and operation for putting the method of the present invention into practice are performed by selecting and re-selecting insert fragments isolated or amplified from, for example, a genome clone or a cDNA clone, which is cloned from a genomic sample and, if necessary, screened and repopulated. The nucleic acid source used in the method of the present invention includes, for example, the genome or cDNA library contained in the mammalian artificial chromosome (MAC), for example, see U.S. Patent Nos. 5,721,118, 6,025,155; for human artificial chromosomes, for example, see Rosenfeld (1997)<i>Nat.Genet.</i>15: 333-335; Yeast artificial chromosome (YAC); Bacterial artificial chromosome (BAC); P1 artificial chromosome, see, for example, Woon (1998)<i>Genomics</i>50: 306-316; vector derived from P1 (PAC), see for example Kern (1997)<i>Biotechniques</i>23: 120-124; mucilage, recombinant virus, bacteriophage or plastid.

The nucleic acids and polypeptides of the present invention can be bound to a solid support (for example) for use in screening and diagnostic methods. The solid support can include, for example, cell membranes (e.g., nitrocellulose or nylon), microtiter plates (e.g., PVC, polypropylene, or polystyrene), test tubes (glass or plastic), measuring rods (e.g., glass, PVC, polypropylene, etc.) Polystyrene, latex and the like), microcentrifuge tubes or glass, silica, plastic, metal or polymer beads or other substrates such as paper. A solid support uses a column containing a metal (such as cobalt or nickel), which is specifically combined with a histidine tag designed on the peptide.

The adhesion of the molecule to the solid support can be direct (that is, the molecule is in contact with the solid support) or indirect (the "linking agent" is bound to the support and the related molecule is bound to the linking agent). Can be covalently (e.g., using a single reactive thiol group of cysteine residues (see, e.g., Colliuod (1993))<i>Bioconjugate Chem.</i>4:528-536)) or non-covalently but specifically (e.g. via immobilized antibodies (see for example Schuhmann (1991))<i>Adv.Mater.</i>3:388-391; Lu (1995)<i>Anal.Chem.</i>67:83-87); biotin/streptavidin system (see, for example, Iwane (1997)<i>Biophys.Biochem.Res.</i>Comm. 230: 76-80); metal chelating membranes, such as Langmuir-Blodgett membranes (see, for example, Ng (1995)<i>Langmuir</i>11:4048-55); self-assembled monolayer of chelated metal (see for example Sigal (1996)<i>Anal.Chem.</i>68: 490-497)) immobilize molecules for polyhistamine fusion.

Indirect bonding can be achieved with various commercially available linking agents. The reactive end can be any of a variety of functional groups, including (but not limited to): amine reactive end, such as N-hydroxysuccinimidyl (NHS) active ester, iminoyl ester, aldehyde, epoxide, Sulfonyl halides, isocyanates, isothiocyanates, and nitroaryl halides; and sulfur reactive ends, such as pyridine disulfide, maleimide, thiophthalimide, and active halogen. Heterobifunctional crosslinking reagents have two different reactive ends, such as amine reactive ends and thiol reactive ends, while the same bifunctional reagents have two similar reactive ends, for example, the bifunctional reagent that allows crosslinking of hydrogen-containing sulfide-containing compounds. Maleimide hexane (BMH). Spacers can have different lengths and be aliphatic or aromatic. Examples of commercially available homobifunctional crosslinking reagents include (but are not limited to) imine esters, such as dimethyl adipimidate dihydrochloride (DMA), dimethyl pimelate Dihydrochloride (DMP) and dimethyl suberimidate dihydrochloride (DMS). Heterobifunctional reagents include commercially available active halogen-NHS active ester coupling agents, such as N-succinimidyl bromoacetate and N-succinimidyl (4-iodoacetyl) aminobenzene Formate (SIAB) and sulfosuccinimidyl derivatives, such as sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB) (Pierce). Another group of coupling agents are heterobifunctional and thiol cleavable agents, such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Pierce Chemicals, Rockford) ,IL).

Antibodies can also be used to bind the polypeptides and peptides of the invention to solid supports. This can be achieved directly by binding a peptide-specific antibody to the column, or by creating a sequence that contains a link to, for example, a known epitope (such as a tag (such as FLAG, myc) or an appropriate immunoglobulin constant domain sequence). The fusion protein chimera of the peptide containing the motif ("immunoadhesin", see, for example, Capon (1989)<i>Nature</i>377:525-531 (1989)) achieve this goal.

The compounds of the invention can be immobilized or applied to an array. The array can be used to screen or monitor the ability of a library of compositions (such as small molecules, antibodies, nucleic acids, etc.) to bind to or modulate the activity of the nucleic acids or polypeptides of the present invention. For example, in one aspect of the present invention, the monitored parameter is the transcriptional performance of the gene containing the nucleic acid of the present invention. By hybridizing samples containing cell transcripts or nucleic acids representing or complementary to cell transcripts (by hybridizing to immobilized nucleic acids on an array or "biochip"), one or more or all of the cell transcripts can be measured . By using nucleic acid "arrays" on microchips, some or all of the cellular transcripts can be quantified at the same time. Alternatively, an array containing genomic nucleic acid can also be used to determine the genotype of a newly designed strain produced by the method of the present invention. "Peptide array" can also be used to quantify multiple proteins at the same time.

The term "array" or "microarray" or "biochip" or "chip" as used herein refers to a plurality of target elements, and each target element includes a defined amount of one or more polypeptides fixed to a defined area on the surface of a substrate (Including antibodies) or nucleic acids. In the practice of the method of the present invention, any known array can be incorporated in whole or in part and/or the method of manufacturing and using the array or its variants, as disclosed in the following patents: for example, US Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; /09217; WO 97/46313; WO 96/17958; see also, for example, Johnston (1998)<i>Curr.Biol.</i>8: R171-R174; Schummer (1997)<i>Biotechniques</i>23: 1087-1092; Kern (1997)<i>Biotechniques</i>23: 120-124; Solinas-Toldo (1997)<i>Genes,Chromosomes &amp; Cancer</i>20: 399-407; Bowtell (1999)<i>Nature Genetics Supp.</i>21:25-32. See also, Published US Patent Application Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.

<b>Host cell and transformed cell</b>The present invention also provides transformed cells comprising a nucleic acid sequence of the present invention (for example, a sequence encoding a polypeptide of the present invention or a vector of the present invention). The host cell may be any host cell familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Exemplary bacterial cells include different species of Escherichia coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and Pseudomonas aeruginosa, Streptomyces and Staphylococcus. Exemplary insect cells include<i>Drosophila</i>S2 and<i>Spodoptera</i>Sf9. Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. Those who are familiar with this technique have the ability to choose an appropriate host.

The vector can be introduced into the host cell by any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun, or Ti-regulated gene transfer. Specific methods include calcium phosphate transfection, DEAE-dextran regulated transfection, lipofection or electroporation.

The designed host cell can be cultured in a conventional nutrient medium that has been modified to activate promoters, select transformants, or amplify the genes of the present invention. After the transformation of a suitable host strain and the growth of the host strain to an appropriate cell density, the selected promoter can be induced in an appropriate manner (such as temperature changes or chemical induction), and the cells can be cultured for an additional period of time to produce the desired polypeptide or its Fragment.

The cells are collected by centrifugation, broken physically or chemically, and the resulting crude extract is retained for further purification. The microbial cells used for protein expression can be disrupted by any conventional method, such methods include freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents. Those skilled in the art are familiar with these methods. The expressed polypeptides or fragments from recombinant cell culture can be recovered and purified by the following methods: including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction Action chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. If necessary, use a protein refolding step in the completed polypeptide configuration. If necessary, high performance liquid chromatography (HPLC) can be used for the final purification step.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the monkey nephrocyte COS-7 line and other cell lines capable of expressing proteins from compatible carriers, such as C127, 3T3, CHO, HeLa and BHK cell lines.

The construct in the host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Depending on the host used in the recombinant production procedure, the polypeptide produced by the host cell containing the vector can be glycosylated or non-glycosylated. The polypeptide of the present invention may or may not include the initial methionine amino acid residue.

A cell-free translation system can also be used to produce the polypeptide of the present invention. A cell-free translation system can use mRNA transcribed from a DNA construct containing a promoter operably linked to a nucleic acid encoding a polypeptide or fragment thereof. In some aspects, the DNA construct can be linearized before performing the in vitro transcription reaction. The transcribed mRNA is then incubated with a suitable cell-free translational extract (such as rabbit reticulocyte extract) to produce the desired polypeptide or fragments thereof.

The expression vector may contain one or more selectable marker genes to provide phenotypic characteristics for the selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as Resistance to tetracycline or ampicillin in Escherichia coli.

For transient expression in mammalian cells, the cDNA encoding the relevant polypeptide can be incorporated into a mammalian expression vector, such as pcDNA1, which is commercially available from Invitrogen Corporation (San Diego, Calif., USA; catalog number: V490-20 ). The ratio is a versatile 4.2 kb plastid vector, which is designed for cDNA expression in eukaryotic systems and cDNA analysis in prokaryotes. CMV promoters and enhancers, splicing fragments, and poly SV40 and polyoma virus sources for adenylation signal, replication, and M13 sources to rescue single-stranded DNA for sequencing and mutagenesis, Sp6 and T7 RNA promoters for generating sense and antisense RNA transcripts And the source of Col E1 high complex essence. Position the multiple restriction site linker properly downstream of the CMV promoter (and 3'of the T7 promoter).

The cDNA insert can be first released from the above-mentioned phagemid incorporated in the pcDNAI multi-enzyme cut-point adapter at an appropriately defined site. The sequencing of the crossover points can be performed to confirm the proper insertion position in pcDNAI. The resulting plastids can then be introduced into a selected mammalian cell host (for example, fibroblasts such as the COS-1 line derived from monkeys (purchased from American Type Culture Collection, Rockville, Md. as ATCC CRL 1650)) for use in Short performance. .

For example, for the transient expression of DNA encoding protein, according to the procedure described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, pages 16.30-16.37, you can borrow DEAE-regulated DNA transfection at every 10⁶About 8μg of DNA in each COS cell was transfected into COS-1 cell and treated with chloroquinine. An exemplary method is as follows. In short, with a density of 5×10⁶COS-1 cells were seeded on each cell/plate, and then grown in DMEM/F12 medium supplemented with FBS for 24 hours. The medium was then removed, and the cells were washed in PBS and then in the medium. Then, a transfection solution containing DEAE dextran (0.4 mg/ml), 100 μM chloroquinine, 10% NuSerum, and DNA (0.4 mg/ml) in DMEM/F12 medium was applied to the cells in a volume of 10 ml. After incubating for 3 hours at 37°C, the cells were washed in PBS and medium as just described, and then shocked with 10% DMSO in DMEM/F12 medium for 1 minute. The cells were grown in a medium supplemented with 10% FBS for 2-3 days, and the plate was placed on ice at the end of the incubation, washed with ice-cold PBS and then removed by scraping. The cells were then collected by centrifugation at 1000 rpm for 10 minutes, and the cell pellets were frozen in liquid nitrogen for later use in protein expression. Northern blot analysis of thawed aliquots of frozen cells can be used to confirm the performance of the cDNA encoding the receptor in the stored cells.

In a similar manner, for example, using two different cell types CHO K1 and CHO Pro5 as hosts can also prepare stably transfected cell lines. In order to construct these cell lines, the cDNA encoding for the relevant protein can be incorporated into the mammalian expression vector pRC/CMV (Invitrogen), which enables stable expression. The insertion at this site places the cDNA under the expression control of the cytomegalovirus promoter and the polyadenylation site and terminator upstream of the bovine growth hormone gene, and allows the cDNA to enter the neomycin resistance gene (by SV40). Early promoter drive) is used as a carrier background for selectable markers.

An exemplary procedure for introducing the plastid constructed as described above is as follows. First, in the MEM medium supplemented with 10% FBS at a density of 5×10⁵Inoculate host CHO cells. After 24 hours of growth, fresh medium was added to the dish, and three hours later, the cells were transfected using a calcium phosphate-DNA co-precipitation procedure (Sambrook et al., supra). Briefly, 3 μg DNA was mixed and incubated with a buffered calcium solution for 10 minutes at room temperature. An equal volume of buffered phosphate solution was added and the suspension was incubated for 15 minutes at room temperature. Then, the incubated suspension was applied to the cells for 4 hours, removed and the cells were shocked with a medium containing 15% glycerol. After three minutes, the cells were washed with medium and incubated for 24 hours under normal growth conditions. Cells resistant to neomycin were selected in α-MEM medium supplemented with 10% FBS containing G418 (1 mg/ml). After about 2-3 weeks, separate individual colonies of resistant G418 cells, select them for pure lineage, and then multiply them for testing purposes.

<b>Instance</b>

The following describes examples involved in the present invention. In most cases, alternative technologies can also be used. The purpose of these examples is to illustrate, and is not to limit the scope of the present invention.

<b>Example 1: Synthesis of intermediate dimethyl-(1-triisopropylsilyl-1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-amine (6)</b><chemistry general="n"><img file="TW200616632A_D0016.tif" /></chemistry>

Step-1- Synthesis of dimethyl-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-amine (<b>2</b>) Add isopropanol (320.0 mL) to a 3-neck round bottom flask, followed by 1H-pyrrole[2,3-b]pyridine<b>1</b>(7-azaindole, 7.10 g, 60.1 mmol), dimethylamine hydrochloride (5.4 g, 0.066 mol) and formaldehyde (2.0 g, 0.066 mol). The reaction mixture was stirred at room temperature for 12 hours and then refluxed for 30 minutes. The suspension solution was evaporated to dryness in vacuum. Add water (60.0 mL) and concentrated hydrochloric acid (6.0 mL) to the residue. The aqueous layer was extracted with ether, and the aqueous layer was neutralized with potassium carbonate. The aqueous layer was extracted with dichloromethane, dried over sodium sulfate and concentrated to produce the product, then further washed with ether and dried to provide the product as a white solid<b>2</b>(7.1 g, yield = 67.4%).

Step-2- Synthesis of dimethyl-(1-triisopropylsilyl-1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-amine (<b>4</b>) Add 7-azamethaine to a round bottom flask<b>2</b>(5.38 g, 30.7 mmol) and N,N-dimethylformamide (25.0 mL) and sodium hydride (1.35 g, 33.8 mmol). Triisopropylsilane chloride (6.8 mL, 32 mmol) was added to the reaction. The reaction was stirred at 20 degrees Celsius for 12 hours. The reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, concentrated and purified with Biotage to give the compound as a colorless oil<b>4</b>(6.0 g, yield = 58.8%).

Step-3- Synthesis of 3-chloromethyl-1-triisopropylsilyl-1H-pyrrole[2,3-b]pyridine(<b>5</b>) Add compound to round bottom flask under nitrogen atmosphere<b>4</b>(500.0 mg, 1.51 mmol) and toluene (5.0 mL). 1.0 M isopropyl chloroformate in toluene (1.6 mL) was slowly added to the reaction mixture at room temperature. Stir the reaction mixture for an additional 2 hours to generate the desired compound<b>5</b>, Which was used in the next step without purification.

Step-4- Synthesis of 3-(6-chloro-pyridine-3-ylmethyl)-1-triisopropylsilyl-1H-pyrrole[2,3-b]pyridine(<b>6</b>, Where X = Cl) in a round-bottomed flask at -40 degrees Celsius under a nitrogen atmosphere, add 5-iodine-2-chloro-pyridine (315.0 mg, 1.32 mmol) or 5-iodo-2-bromo-pyridine and tetrahydrofuran ( 12.0 mL, 0.15 mol). To this reaction was added 2.0 M isopropylmagnesium chloride in tetrahydrofuran (0.72 mL, 1.44 mmol). The reaction mixture was stirred for 40 minutes at -40 degrees Celsius. TLC (hexane/ethyl acetate 2:1) indicated no starting material. 0.6 M CuCN.2LiCl in tetrahydrofuran (2.4 mL, 1.44 mmol) was added to the reaction mixture. The reaction mixture was allowed to go through 5 minutes at room temperature, and trimethyl phosphite (0.29 mL, 2.4 mmol) was added. After 10 minutes, this solution was added containing compound 5 (315.0 mg, from the corresponding grass base<b>4</b>(323 mg, 0.98 mmol) prepared in situ) and toluene (8.0 mL) in a round bottom flask. The reaction was stirred at 20 degrees Celsius for 40 hours. The reaction mixture was poured into water, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, concentrated and purified with Biotage (dichloromethane/methanol 1:10) to give the product as a white solid<b>6</b>, Where X = Cl (230 mg, yield = 59.0%). The reaction conditions, processing procedures, and purification of compound 6 (where X=Br) are the same as the reaction conditions, processing procedures, and purification of compound 6 (where X=Cl).

<b>Example 2: Synthesis of intermediate (6-chloro-pyridine-3-yl)-(1H-pyrrolyl[2,3-b]pyridine-3-yl)-methanone (7)</b><chemistry general="n"><img file="TW200616632A_D0017.tif" /></chemistry>

Add aluminum trichloride (16.0 g, 0.12 mol) and dichloromethane (100.0 mL) to a round bottom flask under a nitrogen atmosphere. Add 1H-pyrrole[2,3-b]pyridine in dichloromethane (20.0 mL) to the reaction mixture<b>1</b>(3.2 g, 0.027 mol). The reaction was stirred at room temperature for 70.0 minutes, followed by the addition of 6-chloropyridine-3-carbon chloride 8 (5.4 g, 0.031 mol) in dichloromethane (10.0 mL). The reaction mixture was stirred at room temperature for 3 hours. Methanol (10 mL) was added to the reaction mixture, and the solvent was evaporated in vacuo. The residue was poured into water, and the precipitated product was removed by filtration. The aqueous layer was extracted with ethyl acetate, and then the organic layer was dried, concentrated and combined with the solid separated by filtration to produce a white solid (M+1=258)<b>7</b>(6.2 g, yield=88.6%).

<b>Example 3: Synthesis of benzyl-[5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-amine (9)</b><chemistry general="n"><img file="TW200616632A_D0018.tif" /></chemistry>

Step-1- Synthesis of benzyl-[5-(1-triisopropylsilyl-1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-amine (10) Add compound to round bottom flask under nitrogen atmosphere<b>6</b>(160.0 mg, 0.40 mmol), benzylamine (0.1 mL, 0.90 mmol), palladium acetate (17.0 mg, 0.076 mmol), toluene (10.0 mL), potassium tert-butoxide (80.0 mg, 0.71 mmol) and 2 -(Di-tertiary butylphosphino)biphenyl (31.4 mg, 0.11 mmol). The reaction was stirred for 3 hours under reflux. TLC and MS indicated no starting material. The reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, concentrated and purified with Biotage (dichloromethane/methanol 1:20) to give the product as a white solid (M+1=471)<b>10</b>(110 mg, yield=58.5%). .

Step-2- Synthesis of benzyl-[5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-amine (9) was added to the round bottom flask Compound<b>10</b>(400.0 mg, 0.85 mmol), tetrahydrofuran (20.0 mL) and tetra-n-butylammonium fluoride (240 mg, 0.93 mmol). The reaction mixture was stirred at 20 degrees Celsius for 30 minutes. TLC indicates no starting material. The reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, concentrated and purified with Biotage (dichloromethane/methanol 1:10) to give product 9 (220 mg, yield=82.4%) as a white solid (M+1=315).

<b>Example 4: Synthesis of [5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-(4-trifluoromethyl-benzyl)-amine ( 12) (Compound 1-1, Table 1)</b><chemistry general="n"><img file="TW200616632A_D0019.tif" /></chemistry>

Step-1- Synthesis of (1H-pyrrolyl[2,3-b]pyridine-3-yl)-[6-(4-trifluoromethyl-benzylamino)-pyridine-3-yl]-methan Ketone (13) is added to the pressure flask<b>7</b>(3.5 g, 0.014 mol) and 4-(trifluoromethyl)benzylamine (9.0 g, 0.051 mol) and tetrahydrofuran (30.0 mL, 0.37 mol) and palladium acetate (200.0 mg, 0.890 mmol) and 2-( Di-tert-butylphosphino)biphenyl (200.0 mg, 0.67 mmol). The reaction mixture was stirred at 180 degrees Celsius overnight, poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated. Add acetic acid (15.0 mL) and H to the residue₂O (5.0 mL). The reaction mixture was stirred at 100 degrees Celsius for 5 hours, and it was concentrated to remove acetic acid. Then use water-containing Na₂HCO₃The residue was treated and extracted with ethyl acetate. The organic layer was washed, dried, concentrated and purified to produce the product as a pale yellow solid (M+1=397)<b>13</b>(1.0 g, yield = 18.5%).

Step-2- Synthesis (1H-pyrrolyl[2,3-b]pyridine-3-yl)-[6-(4-trifluoromethyl-benzylamino)-pyridine-3-yl]-methanol (14) Add compound to the round bottom flask<b>13</b>(210.0 mg, 0.53 mmol) and sodium tetrahydroborate (80.0 mg, 2.11 mmol), and dissolved in N,N-dimethylformamide (5.0 mL) and ethanol (20.0 mL). The reaction was stirred at room temperature overnight, poured into water and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, concentrated and purified with Biotage (dichloromethane/methanol 1:20) to give the product as a white solid (M+1=399)<b>14</b>(63 mg, yield = 30%).

Step-3- Synthesis [5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-(4-trifluoromethyl-benzyl)- amine(<b>12</b>) Add compound to the round bottom flask<b>14</b>(200.0 mg, 0.50 mmol) and trifluoroacetic acid (5.0 mL, 0.065 mol) and triethylsilane (3.0 mL, 0.019 mol). The reaction was stirred at room temperature for 30 minutes, poured into aqueous sodium bicarbonate, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, concentrated and purified to give pure product as a white solid (M+1=383)<b>12</b>(Compound 1-1 in Table 1) (120.0 mg, yield = 62.8%).

<b>Example 5: Synthesis of (4-chloro-benzyl)-[5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-amine (16)( Compound 1-2, Table 1)</b><chemistry general="n"><img file="TW200616632A_D0020.tif" /></chemistry>

Use compound as shown in flow-3<b>6</b>(Where X=Br) is used as the starting material and 4-chlorobenzylamine is used instead of benzylamine to synthesize the compound<b>16</b>(Compound 1-2 in Table 1) (M=348.8).

<b>Example 6: Synthesis of (4-fluoro-benzyl)-[5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-amine (17)( Compounds 1-3, Table 1)</b><chemistry general="n"><img file="TW200616632A_D0021.tif" /></chemistry>

Use compound as shown in flow-3<b>6</b>(Where X=Br) is used as the starting material and 4-fluorobenzylamine is used instead of benzylamine to synthesize the compound<b>17</b>(Compounds 1-3 in Table 1) (M=332.4).

<b>Example 7: Synthesis of (4-methyl-benzyl)-[5-(1H-pyrrolyl[2,3-b]pyridine-3-ylmethyl)-pyridine-2-yl]-amine (18) (Compounds 1-4, Table 1)</b><chemistry general="n"><img file="TW200616632A_D0022.tif" /></chemistry>

Use compound as shown in flow-3<b>6</b>(Where X=Br) is used as the starting material and 4-methylbenzylamine is used instead of benzylamine to synthesize the compound<b>18</b>(Compounds 1-4 in Table 1) (M=328.4).

<b>Example 8: c-kit kinase domain and construction of c-kit sequence</b>The C-kit cDNA sequence (for example) can be obtained from NCBI as GenBank accession number NM_000222. This sequence can be used to clone the c-kit DNA sequence from a commercially available library (such as a cDNA library) or it can be synthesized by a conventional selection method.

The conventional cloning method is used to prepare constructs encoding three c-kit polypeptides, and use them to express c-kit kinase domain polypeptides. A sequence of the active c-kit kinase domain includes residues P551-S948, in which residues Q694-T753 are deleted.

<b>Example 9: Expression and purification of c-kit kinase domain</b>The purified c-kit kinase domain can be obtained by using conventional performance and purification methods. Exemplary methods are described in, for example, Lipson et al., US20040002534 (US application 10/600,868, filed on June 23, 2003), which is incorporated herein by reference in its entirety.

<b>Example 10: Combination verification</b>Determination of IC of compounds by competitive binding assay₅₀(Note: K_IIs the dissociation constant of inhibitor binding; K_DIs the dissociation constant of matrix binding). For this system, according to the following formula, IC₅₀, Inhibitor binding constant and matrix binding constant can be correlated: when using a radioactive isotope-labeled matrix<maths><img file="TW200616632A_D0023.tif" /></maths>IC in the presence of a small amount of labeled matrix₅₀~K_I。

<b>Example 11: c-kit activity test</b>The effects of potential modulators of the kinase activity of c-kit and other kinases can be measured by a variety of different assays known in the art, such as biochemical assays, cell-based assays, and in vivo tests (such as model system tests). These in vitro and/or in vivo assays and tests can be used in the present invention.

In an exemplary biochemical assay, c-kit kinase activity can be determined in the following assay format:<b>Exemplary Biochemical Test</b>Determine the IC related to the inhibition of c-kit kinase activity₅₀Value, where the inhibition of peptide matrix phosphorylation as a function of compound concentration is measured. The compounds in Table 1<b>1－1、1－2、1－3</b>and<b>1－4</b>Dissolve in DMSO to a concentration of 20 mM. Dilute these solutions from 30 μl to 120 μl DMSO (4 mM) and add 1 μl to the calibration plate. Then serially 1:3 dilution (50μl to 100μl DMSO) of these solutions, a total of 8% dilution. Mix the pan vigorously for 10 seconds after each dilution. The diluted sample is then distributed as 1 μl aliquots to the verification plate. Add 8μl substrate (Biotin-(E₄Y)₃, Open Source Biotech, Inc., 0.2 mg/ml in DMSO), PE alpha PY20 (acceptor) and streptavidin (donor) beads (PY20 AlphaScreening kit, Perkin Elmer Life Science Inc. catalog# 676601M) was mixed with 5.5 ml of kinase buffer (50 mM HEPES, pH 7.2, 5 mM MgCl₂, 5 mM MnCl₂, 0.1% NP-40, 50μg/ml BSA). The c-kit kinase domain (starting amino acid M551) prepared by expressing plastid P1332 (pET-N6 BI-PTP and bicistronic PTP with N-terminal non-cleavable His tag) in E. coli And terminating amino acid K949) was added to the solution and mixed. Distribute this in a polypropylene dish with 50μl per well, and then transfer (10μl) to each well of the verification plate. Shake the plate for 20 seconds to mix. The final concentration of c-kit is 50 ng/well. Dilute ATP (100 mM stock solution) from 1 μl to 5 ml kinase buffer, mix the solution thoroughly, transfer 50 μl per well to a polypropylene plate, and then transfer 10 μl per well to a verification plate (final ATP 10 μM). Shake the plate for 30 seconds, and then incubate at 30°C for 30 minutes. Add stop buffer (50 mM EDTA in kinase buffer) at 5 μl per well, and incubate the assay for 30 minutes at room temperature, and then read on the AlphaQuest reader. The phosphorylated matrix causes the PY20 antibody to bind to the acceptor bead and the donor to the acceptor bead to associate the signal with the kinase activity. Use signal to compare compound concentration to determine Ic₅₀. Similarly, for detection³³Compounds listed in Table 1 for radioisotope-labeled ATP verification<b>1－1、1－2、1－3</b>and<b>1－4</b>(Upstate USA, Charlottesville, VA). As measured by at least one of these assays, the IC of all compounds₅₀All are below 1μM.

<b>Additional biochemical and cell-based tests</b>Generally speaking, any protein kinase assay can be adapted to c-kit. For example, in the present invention, assays (e.g., biochemical and cell-based assays) described in Lipson et al., US Patent Publication 2004002534 (incorporated in its entirety by reference) can be used in the present invention.

As an example, the M-07e cell line (DSMZ catalog #ACC 104) is stimulated by SCF that binds to and activates the c-kit tyrosine kinase receptor. The c-kit inhibitor reduces or eliminates the activation of kinases regulated by SCF, resulting in a decrease in cell proliferation of cells stimulated by SCF. Measure the inhibitory effect from the effect of compound concentration on cell growth to evaluate IC₅₀value. Take 5×10 per hole in 96-well filter disc⁴M-07e cells were seeded in 50 μl of Iscove's Medium 1X (MOD, CellGro Mediatech catalog #15-016-CV) supplemented with 10% FBS (HyClone catalog #SH30071.03). The compounds in Table 1<b>1－1</b>and<b>1－2</b>Dissolve in 0.1 mM DMSO and continuously dilute 1:3 for a total of 8%, and add it to the cells to make the final concentration in 100μl cell culture medium (final concentration 0.8% DMSO) is 1, 0.33 , 0.11, 0.037, 0.012, 0.0041, 0.0014 and 0.00046 μM. The cells were also treated with staurosporine as a positive control. Stimulate the cells by adding 20μl of 600 ng/ml SCF to the cell culture medium to a final concentration of 100 ng/ml (Biosource International SCF Kit ligand catalog #PHC2115). At 37°C, 5% CO₂The cells were incubated for three days. Equilibrate the CellTiter-Glo Buffer (Promega Cell Viability Assay catalog #G7573) and substrate to room temperature, and rebuild the enzyme/substrate to reconstitute firefly luciferase/beetle luciferin. Equilibrate the cell plate to room temperature for 30 minutes, and then dissolve it by adding an equal volume of Celltiter-Glo reagent. The dish was mixed on a dish shaker for 2 minutes and then incubated at room temperature for 10 minutes. Read the plate on the Victor Wallac II using the modified luminescence protocol, and read 0.1 second per hole. Luminescence readings assess ATP content, which is directly related to cell number, to use readings as a function of compound concentration to determine IC₅₀value. IC of two compounds₅₀All are below 1μM.

This cell-based assay is also used to assess phosphorylation. Prepare the sample as described in the growth inhibition assay, in which only M-07e is 2×10 per well⁵Cells were seeded in 96-well filter discs. The cells were incubated with the above compound at 37°C for 1 hour, and then the cells were stimulated by adding SCF to a final concentration of 50 ng/ml, and incubated at 37°C for 10 minutes. The medium was removed by centrifugation, and by adding 30μl of lysis buffer (25 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X100, 5 mM NaF, 1 mM sodium vanadate, 10 mM β-glycerol Phosphate, no EDTA (Boehringer-Roche catalog #1873580) to lyse the cells, and keep them on ice for 30 minutes. Take out a 15μl aliquot of the lysate and use Biosource Immunoassay Kit: Human c-kit[pY823] (content# KHO0401), the assay was performed by diluting an aliquot with 85 μl of dilution buffer in the assay disk, incubating for 2 hours at room temperature, and washing the disk 4 times with washing buffer. The detection antibody (100 μl) was added to the dish and the sample was incubated at room temperature for 1 hour, followed by washing 4 times with washing buffer. HRP anti-rabbit antibody (100 μl) was added, and the sample was incubated at room temperature for 30 minutes, followed by washing 4 times with washing buffer. Add stabilized chromogen (100 μl), and incubate the sample at room temperature for 15-25 minutes, then wash 4 times with washing buffer. Add stop solution (100 μl), and read the sample at 450 nm on the Wallac Victor reader. Plot the absorbance of the compound concentration and determine the IC₅₀concentration. IC of two compounds₅₀All are below 1μM.

<b>In vivo model system test</b>For in vivo testing, a suitable animal model system can be selected. For example, for multiple sclerosis, experimental allergic encephalomyelitis (EAE) in rodents is commonly used. This system is well known to us and in (for example) Steinman,<i>Cell</i>1996,<b>85</b>: 299-302 and Secor et al.,<i>J Exp.Med</i>2000,<b>5</b>: 813-821 (the full text of which is incorporated into this article by reference).

Similarly, other model systems can be selected and used in the present invention.

All patents and other references cited in this specification indicate the proficiency of those who are familiar with the present invention, and their full text is incorporated herein by reference, including any tables and figures, to the extent that the references have been individually incorporated The full text of each reference is incorporated by reference.

Those skilled in the art should easily understand that the present invention can be fully adapted to obtain the results and benefits inherent in the foregoing. The methods, variations, and compositions described herein that currently represent preferred embodiments are exemplary and are not intended to limit the scope of the present invention. Those who are familiar with this technology should understand the changes and other uses, which are covered by the spirit of the present invention defined by the scope of the patent application.

It is easy for those familiar with the art to make various substitutions and modifications to the invention disclosed herein without departing from the scope and spirit of the present invention. For example, changes can be made to provide additional compounds of formula I and/or various methods of administration can be used. Therefore, these additional embodiments are within the scope of the present invention and the scope of the following patent applications.

In the absence of any one or more elements or one or more limitations that are not specifically disclosed herein, the invention described illustratively herein can be put into practice. The terms and expressions used are used for description rather than limitation, and the use of these terms and expressions is not intended to exclude any equivalents or parts of the features shown and described, but it should be recognized that the Various modifications are possible within the scope of the invention. Therefore, it should be understood that although the present invention has been specifically disclosed by the preferred embodiments and optional features, those skilled in the art can put into practice the modifications and changes to the concepts disclosed herein, and should be considered as such modifications And the changes are within the scope of the present invention defined by the scope of the attached patent application.

In addition, when describing the features and aspects of the present invention according to the Markush group or other groupings of the alternative, those familiar with the art should realize that the characteristics and aspects of the present invention are also based on the Markush group or other groups. Any individual member or subgroup of members of the group describes the invention.

Also, unless indicated to the contrary, when providing various values for the embodiments, additional embodiments are described by taking any two different values as the end points of the range. These ranges are also within the scope of the above-mentioned invention.

Therefore, the additional embodiments are within the scope of the present invention and the scope of the following patent applications.

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