RU2304436C2 - Using derivatives of n-phenyl-2-pyrimidineamine against mastocyte-base diseases similar to allergic disturbance - Google Patents

Abstract

FIELD: medicine, pharmacology, pharmacy.

SUBSTANCE: invention relates using derivatives of N-phenyl-2-pyridineamide or their salts for preparing a medicinal agent used in treatment of mastocytosis. Also, invention relates to a method for treatment of mammals suffering from mastocytosis. Invention provides enhancing effectiveness of treatment.

EFFECT: valuable medicinal properties of derivatives.

6 cl, 4 tbl, 8 ex

Description

The present invention relates to a new use of derivatives of N-phenyl-2-pyrimidinamine of the formula (I), in which the symbols and substituents have the meaning given below, in free form or in the form of a pharmaceutically acceptable salt, in the preparation of a pharmaceutical composition for the treatment of allergic rhinitis, allergic dermatitis, drug allergy or food allergy, angioedema, urticaria, sudden death syndrome of an apparently healthy child, bronchopulmonary aspergillosis, multiple sclerosis or mastocytosis but; and to a method for treating warm-blooded mammals, preferably a human, in which a therapeutically effective dose of a compound of formula I is administered to a warm-blooded mammal suffering from one of the above diseases.

The present invention relates to the use of N-phenyl-2-pyrimidinamine derivatives of formula I

,

,

Where

R ₁ is 4-pyrazinyl; 1-methyl-1H-pyrrolyl; amino or phenyl substituted with amino lower alkyl wherein the amino group in each case is free, alkylated or acylated; 1H-indolyl- or 1H-imidazolyl attached at the carbon atom of the five-membered ring; or unsubstituted or (ness.) alkyl substituted pyridyl attached at the ring carbon atom and unsubstituted or substituted at the nitrogen atom with oxygen;

R ₂ and R ₃ mean each independently of the other hydrogen or (ness.) Alkyl; one or two of the radicals R ₄ , R ₅ , R ₆ , R ₇ and R ₈ mean each nitro substituted with fluorine (ness.) alkoxy or a radical of the formula II

,

,

Where

R ₉ means hydrogen or (ness.) Alkyl,

X means oxo, thio, imino, N- (ness.) Alkyl, hydroxyimino or O- (ness.) Alkylhydroximino,

Y is oxygen or an NH group,

n is 0 or 1, and

R ₁₀ means an aliphatic radical having at least 5 carbon atoms, or an aromatic, aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or heterocyclic-aliphatic radical, and the remaining radicals

R ₄ , R ₅ , R ₆ , R ₇ and R ₈ mean each independently hydrogen, lower alkyl, which is unsubstituted or substituted by a free or alkylated amino group, piperazinyl, piperidinyl, pyrrolidinyl or morpholinyl, or (lower) alkanoyl, tifluoromethyl, free, ether or ester hydroxyl, free, alkylated or acylated amino group, or

free or ester carboxyl, or salts of such a compound containing at least one salt-forming group,

in order to obtain a medicine for the treatment of allergic rhinitis, allergic dermatitis, drug allergies or food allergies, angioedema, urticaria, sudden death syndrome of an apparently healthy child, bronchopulmonary aspergillosis, mastocytosis or multiple sclerosis.

1-Methyl-1H-pyrrolyl is preferably 1-methyl-1H-pyrrol-2-yl or 1-methyl-1H-pyrrol-3-yl.

Substituted by an amino group or amino (lower) alkyl phenyl R ₁ , in which the amino group is free, alkylated or acylated in each case, means phenyl substituted at any desired position (ortho, meta or para), where the alkylated amino group preferably means mono- or di (ness.) alkylamino, for example, dimethylamino, and (ness.) alkyl part of amino (ness.) alkyl means preferably linear (C ₁ -C ₃ ) alkyl, especially such as methyl or ethyl.

1H-Indolyl attached at the carbon atom of the five-membered ring means 1H-indol-2-yl or 1H-indol-3-yl.

Unsubstituted or substituted (ness.) Alkyl pyridyl attached at the carbon atom of the ring means substituted (ness.) Alkyl or preferably unsubstituted 2-, 4- or preferably 3-pyridyl, for example 3-pyridyl, 2-methyl-3-pyridyl or 4-methyl-3-pyridyl. Pyridyl substituted at the nitrogen atom with oxygen means a radical formed from pyridine N-oxide, i.e. N-oxidopyridyl.

Substituted fluorine (ness.) Alkoxy means (ness.) Alkoxy having at least one, and preferably several fluorine-containing substituents, especially trifluoromethoxy or 1,1,2,2-tetrafluoroethoxy.

When X is oxo, thio, imino, N- (lower) alkylimino, hydroximino or O- (lower) alkylhydroximino, the group C = X means in the above order the radical C = O, C = S, C = NH, C = N- (lower) alkyl, C = N-OH or C = NO- (lower) alkyl, respectively.

Preferably X is oxo.

Preferably n is 0, i.e. group Y is not present.

Y, if present, preferably means an NH group.

The term "lower" in this context means radicals containing up to including up to 7 inclusive, preferably up to 4 inclusive carbon atoms.

(Ness.) Alkyl R ₁ , R ₂ , R ₃ means preferably methyl or ethyl.

An aliphatic radical R ₁₀ containing at least 5 carbon atoms, preferably not more than 22 carbon atoms, usually not more than 10 carbon atoms, means such a substituted or preferably unsubstituted aliphatic hydrocarbon radical, namely such a substituted or preferably unsubstituted alkynyl, an alkenyl or preferably alkyl radical, such as (C ₅ -C ₇ ) alkyl, for example n-pentyl. The aromatic radical R ₁₀ contains up to 20 carbon atoms and means unsubstituted or substituted, for example, in each case unsubstituted or substituted naphthyl, such as, in particular, 2-naphthyl or preferably phenyl, the substituents being preferably selected from cyano, unsubstituted or hydroxy, an amino or 4-methylpiperazinyl substituted lower alkyl such as, in particular methyl, trifluoromethyl, a free, ether or ester hydroxy group, a free, alkylated or acylated amino group and a free or complex ester carboxy groups. In the aromatic-aliphatic radical R _{10, the} aromatic part is as described above, and the aliphatic part is preferably lower alkyl, such as, in particular, (C ₁ -C ₂ ) alkyl, which is substituted or preferably not substituted, for example benzyl. The cycloaliphatic radical R ₁₀ contains mainly up to 30, mainly up to 20 and, in particular, up to 10 carbon atoms, is mono- or polycyclic and means a substituted or preferably unsubstituted radical, for example, such as a cycloalkyl radical, in particular, such as 5 or a 6-membered cycloalkyl radical, such as preferably cyclohexyl. In the cycloaliphatic-aliphatic radical R _{10, the} cycloaliphatic part is as indicated above, and the aliphatic part means preferably lower alkyl, such as, in particular, (C ₁ -C ₂ ) alkyl, which is substituted or preferably not substituted. The heterocyclic radical R ₁₀ contains, in particular, up to 20 carbon atoms and means preferably a saturated or unsaturated monocyclic radical containing 5 or 6 rings and 1-3 heteroatoms, which are preferably selected from nitrogen, oxygen and sulfur, in particular, for example, thienyl or 2-, 3- or 4-pyridyl, or a bi- or tricyclic radical, where, for example, one or two benzene radicals are annelated (condensed) with said monocyclic radical. In the heterocyclic aliphatic radical R _{10, the} heterocyclic moiety is as defined above, and the aliphatic moiety is preferably lower alkyl, such as, in particular, (C ₁ -C ₂ ) alkyl, which is substituted or preferably not substituted.

An esterified hydroxy group is preferably lower alkoxy. An ester hydroxy group preferably means a hydroxyl group esterified with an organic carboxylic acid, such as a lower alkanoic acid, or a mineral acid, such as hydrohalic acid, for example, a halogen mainly such as iodine, bromine or, in particular, fluorine or chlorine.

Alkylamino amino group means, for example, (ness.) Alkylamino group, such as methylamino, or di (ness.) Alkylamino group, such as dimethylamino. An acylated amino group means, for example, a lower alkanoylamino or benzoylamino group.

The esterified carboxyl means, for example, (ness.) Alkoxycarbonyl, such as methoxycarbonyl.

A substituted phenyl radical can have up to 5 substituents, such as fluorine, but especially in the case of relatively large substituents, it is usually substituted with only 1-3 substituents. Examples of substituted phenyl, which may be specifically mentioned, are 4-chlorophenyl, pentafluorophenyl, 2-carboxyphenyl, 2-methoxyphenyl, 4-fluorophenyl, 4-cyanophenyl and 4-methylphenyl.

Salt-forming groups in the compound of formula I mean groups or radicals having basic or acidic properties. Compounds having at least one basic group or at least one basic radical, for example, a free amino group, a pyrazinyl radical or a pyridyl radical, can form acid addition salts with, for example, inorganic acids such as hydrochloric acid, sulfuric acid or phosphoric acid, or with the corresponding organic carboxylic or sulfonic acids, for example, with aliphatic mono- or dicarboxylic acids, such as trifluoroacetic acid, acetic acid, propio new acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or with amino acids such as arginine or lysine, aromatic carboxylic acids such as benzoic acid, 2 -phenoxybenzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid, with aromatic-aliphatic carboxylic acids such as mandelic acid or cinnamic acid, with heteroaromat other carboxylic acids, such as nicotinic acid or isonicotinic acid, with aliphatic sulfonic acids, such as methane, ethane or 2-hydroxyethanesulfonic acid, or with aromatic sulfonic acids, for example, benzene, n-toluene or naphthalene-2- sulfonic acid. In the presence of several main groups, mono- or polyacid addition salts may form,

Compounds of formula I containing acidic groups, for example, a free carboxy group in the radical R ₁₀ , can form metal salts, such as salts with an alkali metal or alkaline earth metal, for example, salts with sodium, potassium, magnesium or calcium, or ammonium salts with ammonia or corresponding organic amines, such as tertiary monoamines, for example triethylamine or tri- (2-hydroxyethyl) amine, or with heterocyclic amines, for example, N-ethylpiperidine or N, N'-dimethylpiperazine.

Compounds of formula I containing both acidic and basic groups can form internal salts.

For the purpose of isolation and purification, as well as in the case of compounds that are further used as intermediates, pharmaceutically unacceptable salts can also be used. However, only pharmaceutically acceptable non-toxic salts are used for therapeutic purposes, and therefore these salts are preferred.

The compounds of formula I can be used in therapy or in the manufacture of medicaments for the treatment of warm-blooded mammals, preferably humans.

The term "allergic rhinitis", as used in context, means any allergic reaction of the nasal mucosa. Such an allergic reaction can occur, for example, constantly, for example, spring conjunctivitis, or temporarily, for example, hay fever.

The term “allergic dermatitis,” as used herein, refers primarily to atopic dermatitis, allergic contact dermatitis and eczematous dermatitis, but also includes, for example, seborrheic dermatitis, lichen planus, urticaria and acne. Atopic dermatitis, as defined in the context, means chronic inflammatory skin lesion observed in an individual with a hereditary predisposition to a reduced threshold for skin itching. Basically, it is characterized by extreme itching, leading to scratching and rubbing, which in turn leads to lesions typical of eczema. Allergic contact dermatitis, as defined in the context, means a form of dermatitis, which is explained by allergic sensitization to various substances that cause inflammatory skin reactions in those who have acquired hypersensitivity to the allergen as a result of previous contact with it.

The term “drug allergy or food allergy” as used herein refers to an allergic reaction caused by a drug or antigens absorbed by food, such as, for example, strawberries, milk or eggs.

The term "bronchopulmonary aspergillosis" refers to infection of the lungs with a fungus of the species Aspergillus.

The term "mastocytosis", as used in context, refers to systemic mastocytosis, for example, mastocytoma, and, in particular, to neoplasms associated with the pathology of mast cells in dogs.

Compounds of formula I are hereinafter referred to collectively as “compounds of the invention”.

Mastocytes play an important role as primary effector cells in the allergic disorders referred to in the context. Antigen-specific IgE-mediated mast cell degranulation leads to the subsequent release of chemical mediators and numerous cytokines and the synthesis of leukotrienes. In addition, mast cells are involved in the pathogenesis of multiple sclerosis.

Mast cell-based neoplasms are found in both humans and animals. In dogs, mast cell-based tumors are called mast cells, and the disease is common, accounting for 7% -21% of dog tumors. A distinction should be made between human mastocytosis, which is transient or painless, and mastocytic neoplasia of dogs, which behaves unpredictably and is often aggressive and metastatic. For example, human single mastocytomas essentially never metastasize; in contrast, 50% of dog mast cells are malignant, as established by Hottendorf and Nielsen (1969) after a review of 46 published tumor reports in 938 dogs.

In a pet population, cancer is a spontaneous disease. Pet owners, encouraged to extend the quality of life of their animals, often seek specialized care and treatment from veterinary oncologists in private veterinary clinics and veterinary clinics at higher medical schools across the country.

The therapeutic possibilities of veterinary patients with malignant tumors are similar to those for humans, including surgery, chemotherapy, radiotherapy and biotherapy. It is estimated that there are 42 million dogs and approximately 20 million cats in the United States. According to preliminary estimates of cancer cases, this diagnosis is first made to approximately 4 million dogs and a similar number of cats each year.

Dog skin mast cells are a common problem. Most mast cells are benign and can be cured by simple resection; however, if they are relapsing or metastasizing to distant sites, then therapeutic options are limited. Treatment options for recurrent lesions may include external targeted radiotherapy. For distant metastases or disseminated disease, the use of protocols including chemotherapy with lomustine® and vincristine has shown some advantage. The location of mast cell metastases includes skin, regional lymph nodes, spleen, liver and bone marrow.

The involvement of the kit receptor in the pathogenesis of mastocytosis is suggested based on the observation that several mutations have been identified in many lines of mast cells leading to constitutive activation of the kit. For example, a point mutation in the human c-kit receptor, which causes Val to be replaced by Asp816 in the phosphotransferase domain and receptor autoactivation, occurs in a long-cultivated line of human mastocytic leukemia (HMC-1) and in the corresponding codon in two rodent mastocyte lines. Moreover, an activating mutation has been found in situ in some cases of human mastocytosis. Two other activating mutations are found in the intracellular kit receptor site adjacent to the membrane, i.e. replacing Val560Gly in the HMC-1 human mast cell line and several amino acid deletions (Thr573-His579) in the rodent mast cell line called FMA3.

It can be shown using generally accepted test models and especially those test models which are described in the context that the compounds of the invention or in each case a pharmaceutically acceptable salt based on this lead to the effective prevention or preferably treatment of at least one of those mentioned in disease context. The person skilled in the art is quite capable of choosing the appropriate test model to prove the therapeutic indications and predominant effects mentioned previously. For example, pharmacological activity can be demonstrated in a clinical study or in an experimental procedure, as essentially described below.

Part a

Example 1

This example demonstrates the in vitro effects of the compounds of the invention on stem cell factor (SCF) dependent evolution of the growth of cultured human mast cells generated by CD34 ⁺ cord blood cells using the culture system described by Kinoshita N., Sawai N., et al., Blood, 1999, 94, 496-508. According to flow cytometric analysis, more than 90% of the isolated cells were positive for the CD34 receptor (CD34 ⁺ ).

Reagents and antibodies

The compounds of the invention were dissolved in PBS at a concentration of 10 ^-2 M and stored at -80 ° C. Fully trans-retinoic acid (Sigma) was dissolved in ethanol at a concentration of 10 ^-2 M and stored in light-protected vials at -80 ° C. Purified tryptase monoclonal antibody (mAb) (MAB1222) is available from Chemicon International Inc., CA. For flow cytometric analysis, monoclonal antibodies to CD34 [8G12, fluorescein isothiocyanate (FITC) as a label] and CD11b [Leu 15, R-phycoerythrin (PE) as a label] were purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA) and mAb k CD41 (SZ22, FITC) from Immunotech SA (Marseilles, France).

Monoclonal antibody to glycophorin A (GPA, JC159, FITC) can be obtained from Dako (Glostrup, Denmark). For Western blotting and immunoprecipitation, monoclonal antibodies to c-kit (NU-c-kit) and to phosphotyrosine (4G10) are available from Nishirel and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively.

Suspension cultures

Serum-free liquid cultures were maintained in 24-well culture plates (# 3047; Becton Dickinson). Twenty thousand CD34-positive cells were cultured in each well containing 2 ml of α-medium supplemented with 1% bovine serum albumin (BSA), fully saturated with human transferrin iron (approximately 98% purity, Sigma), 16 μg / ml soya bean lecithin (Sigma), 9.6 μg / ml cholesterol (Nakalai Chemicals Ltd., Japan) and 20 ng / ml stem cell factor (SCF), 10 ng / ml granulocyte macrophage colony stimulating factor (GM-CSF), 2 u / ml erythropoietin (EPO), 10 ng / ml thrombopoietin oxidase (TPO), various concentrations of the compounds of the invention, od th or in combination. In order to investigate the effects of the compound of the invention on SCF-dependent mast cell development, cells cultured for 10 weeks grown in the presence of 20 ng / ml SCF from CD34-positive umbilical cord blood cells were used as target cells. Cultured for 10 weeks, 5 × 10 ⁴ -10 × 10 ⁴ cells were incubated for 2 weeks in 24-well culture plates containing 20 ng / ml SCF with various concentrations or without a compound of the invention. The plates were incubated at 37 ° C in a humidified atmosphere, purged with a mixture of 5% CO ₂ , 5% O ₂ and 90% N ₂ . After 4 weeks of cultivation, half of the culture medium was replaced every 2 weeks with fresh medium containing factor (s). The number of viable cells was determined using a trypan blue displacement test using a hemocytometer.

Clonal Cell Culture

Mast cell colonies were analyzed in 35 mm Lux dishes for suspension cultures (# 171099; Nunc, IL). The culture consisted of 10-week-old cultured cells (4000 cells / ml) grown in the presence of 10 ng / ml SCF, α-medium, 0.9% methylcellulose (Shinetsu Chemical, Japan), 1% BSA, 300 μg / ml fully saturated human transferrin iron, 16 μg / ml lecithin from soybeans, 9.6 μg / ml cholesterol and 100 ng / ml SCF with or without 10 ^-6 M compounds of the invention. The cups were incubated at 37 ° C in a humidified atmosphere, purged with a mixture of 5% CO ₂ , 5% O ₂ and 90% N ₂ . After 4 weeks of cultivation, aggregates of 30 cells or more were scored as colonies of mast cells, and aggregates containing 10-29 cells as clusters of mast cells. Thirty individual colonies and clusters were removed and stained with anti-tryptase mAb or murine IgGI using the alkaline phosphatase-anti-alkaline phosphatase (ARAAP) technique. Almost all of the cellular components were tryptase positive.

Cytochemical and immunological staining

Cultured cells were distributed over the surface of the slides using Cytospin II. The cytochemical reaction with peroxidase (POC) was carried out in a well-known manner. The reaction with anti-tryptase mAbs was detected using the APAAR method (Dako APAAP Kit System, Dako Corp., CA) as described by F. Ma, K. Koike et al., Brit. J. Hematol. 1998, 100, 427-35.

Immunoprecipitation and Western Blotting

Immunoprecipitation and Western blotting were performed as described by T. Kamijo, K. Koike et al., Leuk. Res. 1997, 21, 1097-106.

Flow cytometric analysis

To analyze surface markers on cultured cells, 1-2 × 10 ⁵ cells were selected in plastic tubes and incubated with appropriately diluted FITC- or PE-mAb, as described by Kinoshita N., Sawai N. et al. Blood 1999, 94, 496- 508. Cells were washed twice, after which their surface markers were analyzed using a FACScan flow cytometer. Viable cells were separated according to their direct light scattering characteristics and side scattering characteristics. The proportion of positive cells was determined by comparison with cells stained with FITC- or PE-conjugated mouse Ig isotype compatible.

Definition of cell apoptosis

The study of cell apoptosis was performed by flow cytometric analysis using propidium iodide (PI, Sigma) in accordance with the method described by N. Sawai, K. Koike and others. Stem Cells. 1999, 17, 45-53. In order to reduce the number of cells undergoing apoptosis, necrosis, or already dead, centrifugation in a percoll gradient can be used. Ten-week-old cultured cells were layered on 27% percoll (Sigma) in α-medium and 54% percoll in PBS. After centrifugation, cells were collected from the surface of the solution with two different concentrations of percoll, washed with PBS and treated with 1 ml of Ortho PermeaFix ^™ for 40 min at room temperature. The cells were then incubated with the DNase-free RNase (Sigma) system for 15 min at 37 ° C and stained with PI for 15 min. The DNA content of 2 × 10 ⁴ cells was monitored using a flow cytometer. Ten-week-old cultured cells (2 × 10) exposed to SCF or SCF and the compounds of the invention were lysed for 10 min on ice in 100 μl of hypotonic lysis buffer [10 mM Tris buffer (pH 7.5), 10 mM ethylenediaminetetraacetic acid ( EDTA), pH 8.0, 0.5% Triton X-100]. After centrifugation for 10 min at 14000 g, the supernatant was transferred to a new tube and treated with 0.2 mg / ml RNase A (Sigma) and 0.2 mg / ml proteinase K (Sigma). DNA was precipitated with 120 μl of isopropanol and 20 μl of 5 M sodium chloride overnight at -20 ° C. After centrifugation at 14000g, the pellets were dried, dissolved in 20 μl Tris-EDTA and then samples were analyzed by gel electrophoresis in 2% agarose and stained with ethidium bromide.

Analysis of histamine, tryptase and cytokine levels

Histamine concentrations in cell lysates obtained by treating cultured cells with a 0.5% non-ionic non-detergent non-P-40 detergent and in the supernatant were measured using a histamine radioimmunoassay kit (RIA) (Immunotech) as described by Kinoshita T., Sawai N. and Dr. Blood 1999, 94,496-508.

Statistical analysis

All experiments had to be carried out three times. To determine the significance of the difference between two independent groups, one could use the unpaired t-test or the Mann-Whitney U-test when the data were not normally distributed.

Results for N- {5- [4- (4-methylpiperazinomethyl) benzoylamido] -2-methylphenyl} -4- (3-pyridyl) -2-pyrimidinamine monomesylate (STI571B)

Adding STI571B at a concentration of 10 ^-6 M or higher in the SCF culture almost completely inhibits progeny generation.

In SCF supplemented with STI571B, the number of viable cells remains unchanged on day 2, and then decreases in a time-dependent manner. On day 14, the total number of cells reaches a negligible level. Compatible with the results obtained on ten-week-old cultured mast cells, STI571B at a concentration of 10 M significantly inhibits cell production by CD34-positive cells upon stimulation with SCF. Moreover, STI571B induces programmed death of cultured mast cells grown under stimulation with SCF. The percentage of subdiploid nuclei increases as the cultivation period increases. This observation can be confirmed by DNA separation in cells exposed to SCF supplemented with STI571B by gel electrophoresis. Compound STI571B exhibits its inhibitory properties in the early phase of development of mast cells, as well as in the growth phase of late mast cells.

The results demonstrate growth inhibition of SCF-dependent human mast cells by a compound of the invention, for example, STI571B. It follows that the compounds of the invention can be used to treat diseases mentioned in the context.

Part B: mastocytosis

Example 2: methods

Reagents: Novartis Pharma; Basel, Switzerland provided salt I for use in these experiments. Fresh 10 mM inhibitor stock solutions were prepared before each experiment, dissolving the compound in 1 ml of phosphate buffered saline (PBS; Gibco-BRL).

Antibodies: a polyclonal rabbit anti-kit antibody (c-kit Ab-1) was used at a dilution of 1: 500 (c-kit Ab-1; Oncogene, Cambridge, MA). The anti-phosphotyrosine antibody (PY20) was used at a 1: 1000 dilution (PY20 Transduction Laboratories; Lexington, KY). A peroxidase-conjugated goat anti-mouse antibody was used at a dilution of 1: 5000 and a goat anti-rabbit antibody at a dilution of 1: 10000 (Pierce; Rockford, IL).

Cell lines: Cells of the BR and C2 lines of dog mast cells were obtained from Dr. George Caughey (University of California at San Francisco, San Francisco, CA). Both cell lines were maintained in Eagle's Dulbecco Modified Medium (DMEM) supplemented with 2% bovine bovine serum albumin from calf serum, 1 mM L-glutamine, 12.5 mM N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid ( HEPES), pH 7.5, 0.25 mg / ml histidine, 1% penicillin-streptomycin and 1% fungicone. BR and C2 cells were derived from canine mast cell tumors and were initially adapted in a long-living culture after initial passage to immunodeficient mice (1; 2). The BR cell line has a point mutation (T1752C), leading to the replacement of leucine with proline at amino acid 575 (located next to the membrane domain). The C2 cell line has an internal tandem doubling (ITD) of the kit site adjacent to the membrane. Translation of this ITD leads to a repeated doubling of amino acid residues at the 3'-end of exon 11 (3; 4).

Proliferation assays: cells were placed in 96-well plates at a density of 40,000 cells / well with normal culture medium and salt I at various concentrations. Proliferation was measured after 48-72 hours using an analysis based on the use of 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide (XTT) sodium salt (Roche Molecular Biochemicals; Indianapolis , IN). (5).

Protein lysates: BR and C2 cells were washed twice in PBS and then left resting in Optimem medium (Gibco-BRL) at 37 ° C for approximately 18 hours. Then, the cells were incubated for 90 minutes in the presence of salt I at various concentrations. After this incubation, the cells were besieged and lysed using 100-250 μl of protein lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate with the addition of inhibitors aprotinin, leipeptin, pepstatin, phenylmethylsulfonyl fluoride (PMSF) and sodium orthovanadate [Sigma]). Analysis by Western immunoblotting was performed as described previously (6).

Example 3: Compound I inhibits a constitutively activated kit kinase. associated with mast cell tumors of dogs

In order to investigate the efficacy of compound I in inhibiting the kinase activity of mutant forms of the dog kit receptor, the applicants used two cell lines (BR and C2) that express two different constitutively activated kit isoforms. In these cells, both kit mutations are located near the membrane domain and are homologous to mutations observed in humans in gastrointestinal stromal tumors (GISTs) (7; 8). Lysates obtained from BR and C2 cells were probed with an antiphosphothyrosine antibody, and kit receptor activation was evaluated by measuring autophosphorylation. As previously reported, autophosphorylation of kit in these cells was observed in the absence of SCF [Steel Factor (SLF) = SCF] (4; 9). Autophosphorylation inhibition of the kit was dose dependent, complete inhibition was observed using doses of 10 and 1.0 μM. Almost complete inhibition was observed using a dose of 0.1 μM. Limited c-kit autophosphorylation was observed using a 0.01-0.01 μM dose of compound I. Thus, compound I not only inhibits the autophosphorylation of the mutated c-kit receptor in these cells, but is also a more potent inhibitor of this mutated receptor than c wild-type kit (IC ₅₀ 100-200 nM) (5). In order to determine whether compound I modulated the expression of kit protein, the membrane was peeled off and again probed with an anti-c-kit antibody. In cells treated with compound I, there was no change in c-kit protein expression. Therefore, compound I attenuates the autophosphorylation of the mutant canine kit polypeptide by inhibiting the kinase activity of kit rather than by downregulating the expression of kit protein.

Example 4: Compound I inhibits the proliferation of cell lines of mast cells of dogs

To study the biological effect of inhibiting the kinase activity of the c-kit mutant receptor, the applicants cultured BR or C2 cells for 48-72 hours in the presence of compound I at various concentrations. At an inhibitor concentration of 0.1-10 μM, proliferation was reduced by 90-95% compared with cells treated only with medium. Partial inhibition of proliferation was observed at doses of 0.001-0.01 μM of compound I. The decrease in proliferation observed at doses of an inhibitor of 0.01-10 μM was statistically significant (p <0.001). Therefore, compound I inhibits the proliferation of BR and C2 cells with a response in the same dose range as that found to inhibit receptor autophosphorylation. Morphological observations of the cells treated with the inhibitor revealed changes corresponding to apoptosis (data not shown).

Table 1:
BR cells Averages % Standard deviation Standard deviation, % Cells 0.929 one hundred 0,030447 3 5 μM 0,083 9 0,001732 0 1 μM 0.105 eleven 0.002 0 0.1 μM 0.105 eleven 0,002082 0 0.01 μM 0.479 52 0,043016 5 0.001 μM 0.781 84 0,033081 four Table 2:
C2 cells Averages % Standard Deviation (SD) Standard deviation, % Cells 1,236 one hundred 0,04417 four 5 μM 0,032 3 0.005686 0 1 μM 0,037 3 0.013868 one 0.1 μM 0,028 2 0,003512 0 0.01 μM 0.754 61 0.185236 fifteen 0.001 μM 1,065 86 0.055245 four

Tables 1 and 2: BR and C2 cells were placed in 96-well plates at a concentration of 40,000 cells / well and cultured in normal growth medium and with a change in the concentration of compound I. Cell proliferation was measured after 72 hours using an XTT-based assay system. Each concentration of compound I was analyzed three times. The results were expressed as the percentage of maximum proliferation (only cells, not compound I) ± 1 standard deviation. Representative results of one of six independent experiments are presented.

Compositions for pets and humans

Example 5: capsules with methanesulfonate 4 - [(4-methyl-1-piperazin-1-ylmethyl) -N- [4-methyl-3 - [[4- (3-pyridinyl) -2-pyrimidinyl] amino] phenyl] β-crystalline benzamide

Capsules containing 11.95 mg of the title compound (identical name salt I) corresponding to 10.0 mg of compound I (free base) as the active substance were prepared as the following composition.

Composition Salt i 11.95 mg Cellulose MK GR 9.2 mg Crospovidone XL 1.5 mg Aerosil 200 0.2 mg Magnesium stearate 0.15 mg 23.0 mg

Capsules were prepared by mixing the components and loading the mixture into hard gelatin capsules, size 1.

Example 6: capsules with methanesulfonate 4 - [(4-methyl-1-piperazin-1-ylmethyl) -N- [4-methyl-3 - [[4- (3-pyridinyl) -2-pyrimidinyl] amino] phenyl] β-crystalline benzamide

Capsules containing 10.0 mg of the compound named in the title (identical name salt I) as the active substance were prepared as the following composition.

Composition Active substance 10.0 mg Avicel 20.0 mg Polyvinyl Polypyrrolidone XL (PVPPXL) 1.5 mg Aerosil 0.2 mg Magnesium stearate 0.15 mg 31.85 mg

Capsules were prepared by mixing the components and loading the mixture into hard gelatin capsules, size 1.

Example 7: capsules with methanesulfonate 4 - [(4-methyl-1-piperazin-1-ylmethyl) -N- [4-methyl-3 - [[4- (3-pyridinyl) -2-pyrimidinyl] amino] phenyl] β-crystalline benzamide

Capsules containing 119.5 mg of the title compound (identical name for the mesylate of compound I) corresponding to 100 mg of compound I (free base) as the active substance were prepared as the following composition.

Composition Mesylate Compound I 119.5 mg Cellulose MK GR 92 mg Crospovidone XL 15 mg Aerosil 200 2 mg Magnesium sulfate 1.5 mg 230 mg

Capsules were prepared by mixing the components and loading the mixture into hard gelatin capsules, size 1.

Example 8: An example of an alleged series of diseases in domestic dogs with measurable skin mast cell tumors.

The studied patients were domestic dogs with measurable and histologically confirmed mast cell tumors. Cases of the disease were limited to those with measurable biopsy lesions.

Acceptable criteria were:

- histologically confirmed measurable cutaneous mast cell tumors,

- cases of diseases in which a serial biopsy with a 2-mm Keyes punch before and during therapy is required,

- histological stage (II - intermediate or III - undifferentiated),

- functional status 0 or 1 (Karnofsky modification - table 3),

- informed consent of the owner.

(a) Exclusion criteria were:

- simultaneous cytotoxic chemotherapy,

- Prednisone and non-steroidal anti-inflammatory drugs may not have begun to act within 30 days of the study; if prednisone or non-steroidal anti-inflammatory drugs were administered long before 30 days, their intake could be continued,

- An abnormal test for serum bile acid (liver function).

Table 3: functional status (Karnovsky modification) Rating Description 0 Normal activity one Limited activity; decreased activity from the condition before the disease 2 A state of moderate severity; the ability to move only for the necessary life; consistently defecate and urinate in suitable areas 3 Incapable; should be forced to take food; unable to limit urination and bowel movements in suitable areas four Dead

The pre-treatment examination in all cases included a complete blood count, leukocyte film, serum biochemical analysis, analysis of urine, serum bile acids (during fasting and after feeding), information on the size of regional lymph nodes, radiographic and ultrasound examinations of the abdominal cavity. The treatment regimen was to administer 25 mg / kg of salt I orally four times a day for 60 days.

Treatment continued in all cases for 60 days if disease progression was not observed. If the experiment achieves a partial or complete response, the continuation of therapy for an additional 60 days can be discussed. It is advisable to re-examine cases of diseases with successful completion of therapy.

The effectiveness of compound I is evaluated by measurable cutaneous mast cell tumors using clinical endpoints. Biological endpoints can be taken from serial biopsy data collected from skin tumors and from blood samples available throughout the course of treatment.

Clinical endpoints include the response rate to treatment of the measured tumors, an objective response against the measured tumor, and the progression time of the measured tumor. All adverse side effects will be recorded.

"Objective responses of tumors", as defined below, are observed in the treatment of compound I and indicate the effectiveness of the therapeutic regimen.

In particular, complete responses and partial responses to treatment with compound I may be observed. In addition, it can be observed that numerous animals receiving treatment show a stable disease, while less treated animals show a progressive disease. It can also be observed that fewer animals receiving treatment show a relapse compared to untreated animals. The period to disease progression, the duration of remission, and survival can be increased by treatment with compound I.

"Complete response (CR)" is defined as the complete disappearance of the clinical confirmation of cancer and any symptom associated with it.

A “Partial Response (PR)” is defined as a 50% or greater decrease in the sum of the results of measurements of representative damage without increasing the size of any damage or the appearance of any new damage.

"Stable disease (SD)" is defined as the lack of response to treatment or a reaction weaker than that determined by a partial response, or the progression of the disease without the appearance of any new lesions or worsening of clinical symptoms.

"Progressive disease (PD)" is defined as a clear increase of at least 50% in the size of any measurable lesion or the appearance of new lesions.

"Relapse (R)" is defined as the occurrence of new lesions or the reoccurrence of old lesions in dogs that had a complete response to treatment; in dogs that had only a partial response, relapse is defined as at least a 50% increase in the sum of the results of measurements of representative damage compared with measurements obtained during the maximum response.

"Time to progression (TTR)" is recorded from day 0 of the protocol. TTR will be defined as the number of days from the start of therapy (from day 0) to relapse (R),

"Duration of remission" is defined as the number of days from the start of an objective response (PR or CR) to relapse.

"Survival" is defined as the number of days from the start of treatment with compound I to death. Cases of death will be noted, but may include the progression of the disease, toxicity, etc.

The results demonstrate the inhibition of mast cells by the compounds of the invention, for example, STI571B.

Preference is given to compounds of the invention, where one or two of the radicals R ₄ , R ₅ , R ₆ , R ₇ and R _{8 are} each nitro or a radical of formula II, where

R ₉ means hydrogen or (ness.) Alkyl,

X is oxo, thio, imino, N- (ness.) Alkylimino, hydroxyimino or O- (ness.) Alkylhydroximino,

Y is oxygen or an NH group,

n is 0 or 1, and

R ₁₀ means an aliphatic radical containing at least 5 carbon atoms, or an aromatic, aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic, heterocyclic or heterocyclic-aliphatic radical, and the remaining radicals

R ₄ , R ₅ , R ₆ , R ₇ and R ₈ mean each independently hydrogen, lower alkyl, which is unsubstituted or substituted by a free or alkylated amino group, piperazinyl, piperidinyl, pyrrolidinyl or morpholinyl, or (lower) alkanoyl, trifluoromethyl, free, ester or ester hydroxy group, free, alkylated or acylated amino group or free or esterified carboxy group, and the remaining substituents are as defined above.

Particular preference is given to compounds of the invention,

Where

R ₁ means pyridyl or N-oxidopyridyl, each of which is associated with a carbon atom,

R ₂ and R ₃ mean each hydrogen,

R ₄ means hydrogen or (ness.) Alkyl,

R ₅ means hydrogen, (ness.) Alkyl or trifluoromethyl,

R ₆ means hydrogen,

R ₇ means nitro, fluoro-substituted (ness.) Alkoxy or a radical of the formula II,

Where

R ₉ means hydrogen,

X means oxo,

n is 0, and

R ₁₀ means pyridyl bonded to a carbon atom, phenyl which is unsubstituted or substituted by halogen, cyano, lower alkoxy, carboxy, lower alkyl or 4-methylpiperazinylmethyl, or (C ₅ -C ₇ ) alkyl, thienyl , 2-naphthyl or cyclohexyl, and

R ₈ means hydrogen.

Particular preference is given to compounds of the invention in which at least one of the radicals R ₃ and R ₈ is lower alkyl and the remaining substituents are as defined above.

Preference is given to all compounds of the invention,

Where

R ₁ means pyridyl bonded to a carbon atom,

R ₂ , R ₃ , R ₅ , R ₆ and R _{8 are} each hydrogen,

R ₄ means (ness.) Alkyl,

R ₇ means a radical of formula II,

Where

R ₉ means hydrogen,

X means oxo,

n is 0, and

R ₁₀ means 4-methylpiperazinylmethyl.

Preference is given to all especially to the compound according to the invention of formula I, which means CGP 57148B {N- {5- [4- (4-methylpiperazino-methyl) benzoylamido] -2-methylphenyl} -4- (3-pyridyl) -2- monomesylate pyrimidinamine}.

Most preferably, the compound of the invention is used in the form of a monomesylate salt.

In one preferred embodiment of the invention, the disease to be treated is selected from allergic rhinitis, allergic dermatitis, drug allergy and food allergy. In another preferred embodiment, the disease to be treated is multiple sclerosis. In a further embodiment of the invention, the disease to be treated is selected from angioedema, urticaria, sudden death syndrome of an apparently healthy child, and bronchopulmonary aspergillosis. In addition, the compounds of the invention can be used to treat systemic mastocytosis, especially mastocytomas. The latter disease is a malignant disease with extensive metastases, especially in dogs.

Thus, the compounds of the invention are particularly useful in the preparation of a medicament for the treatment of dog mast cells.

Compounds according to the invention with respect to generic and specific characteristics are disclosed in applications EP 0564409 A1 and WO 99/03854, especially in the claims concerning the compounds and end products of the working examples, the object of the final products, pharmaceuticals, and thus the claims are included in this application by reference to these publications. Also included are the corresponding stereoisomers, as well as the corresponding polymorphs, for example, the crystalline modifications that are disclosed therein.

EP 0 564 409 A1 discloses that the compounds of the invention are useful in the treatment of cancer, thrombosis, psoriasis, fibrosis, dermatosclerosis and atherosclerosis. In accordance with the present invention, it has been found that the compounds of the invention have beneficial effects on allergic rhinitis, allergic dermatitis, drug allergies or food allergies, angioedema, urticaria, sudden death syndrome of an apparently healthy child, bronchopulmonary aspergillosis, multiple sclerosis and, in addition, mastocytosis , especially mastocytic neoplasms of dogs.

In accordance with the specific findings of the invention, the present invention also provides a method of treating warm-blooded mammals, including humans, in which a therapeutically effective dose of a compound of the invention is administered to such a warm-blooded mammal, preferably a human or a dog, most preferably a human suffering from one of the diseases mentioned in the context .

The invention also relates to a method for administering to a dog object with mast cells a compound of the invention or a pharmaceutically acceptable salt thereof, which comprises administering a pharmaceutically effective amount of a compound of the invention or its pharmaceutically acceptable salt to a dog for a period of time preferably more than 1 month, 2 months, or even 3 months. The invention relates generally to such a method in which a daily dose of salt I of about 20-200 mg, preferably 80-160 mg, in particular 125 mg, is administered.

Preferably, the invention relates to a use or method wherein a daily dose of the 4- (4-methylpiperazin-1-ylmethyl) -N- [4-methyl-3- (4-pyridin-3-yl) pyrimidin-2-ylamino) phenyl monomethanesulfonate salt ] a benzamide of formula I is administered to a dog, and said daily dose contains an amount of said monomethanesulfonate salt sufficient to maintain its plasma level of at least 0.2 μM.

The term "treatment method", as used in the context, refers particularly to the method of preventing diseases mentioned in the context, i.e. prophylactically administering a pharmaceutical composition comprising a compound of the invention to healthy patients in order to prevent an outbreak of diseases referred to in the context.

The present invention also relates to a pharmaceutical composition for treating at least one of the diseases mentioned in a context comprising a compound of the invention. Pharmaceutical compositions for treating at least one of the diseases mentioned in the context include an effective amount of the compounds of the invention along with pharmaceutically acceptable carriers that are suitable for topical, enteral, for example, oral or rectal, or parenteral administration and can be inorganic or organic, solid or liquid. For oral administration, tablets or gelatin capsules are mainly used, including the compounds of the invention along with diluents and / or lubricants, for example silicic acid, talc, stearic acid or its salts and / or polyethylene glycol, but also lotions, gels or creams. Tablets may contain binders, starches, gelatin, methyl cellulose, sodium carboxymethyl cellulose and / or polyvinyl pyrrolidone, disintegrants and / or effervescent mixtures or adsorbents, colorants, flavors and sweeteners. The compounds of the invention may also be used in the form of parenteral formulations or in the form of infusion solutions. Topical administration means, for example, application to the skin. Another form of local administration is administration to the eye, for example, for the treatment of spring conjunctivitis. The pharmaceutical compositions may be sterilized and / or may include excipients, for example, preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for regulating the osmotic pressure and / or buffers. The present pharmaceutical compositions are prepared in a manner known per se, for example, by conventional mixing, granulation, drug, dissolution or lyophilization techniques, and contain about 1-100%, especially about 1-20% of the active ingredient, one or more.

The compounds of the invention can, for example, be formulated as described in Examples 4 and 6 of WO 99/03854.

The dose range of the compounds of the invention to be used depends on factors known to those skilled in the art, including the type of warm-blooded mammal, body weight and age, route of administration, particular substance administered and the disease to be treated. Unless stated otherwise in the context, the compounds of the invention are preferably administered one to four times a day, or immediately, as soon as an allergic reaction occurs. In addition, the compounds of the invention, especially N- {5- [4- (4-methyl-piperazinomethyl) benzoylamido] -2-methylphenyl} -4- (3-pyridyl) -2-pyrimidinamine (STI571B), are preferably administered to warm-blooded mammals , mainly to humans, in a dosage range of about 10-750 mg / day, preferably 30-600 mg / day, more preferably 30-300 mg / day.

In dogs, depending on the breed, age, individual condition, route of administration and the clinical picture in question, effective doses, for example, daily doses of about 20-200 mg, preferably 80-160 mg, in particular 125 mg, are administered to warm-blooded mammals with a mass about 5 kg. For adult dogs weighing about 5 kg with inoperable and / or metastatic malignant canine mast cell neoplasia, an initial dose of 125 mg daily can be recommended. For dogs with an inadequate response, after evaluating the response to therapy with a dose of 125 mg daily, dose escalation can be considered without risk, and dogs can be treated until they benefit from the treatment in the absence of limiting toxicities. Doses can be titrated so as to reach plasma levels of at least 0.2 μM (micromolar level), preferably at least 0.5 μM, more preferably at least 1 μM. Achieving and / or maintaining a plasma level of about 1 μM is particularly preferred.

Claims (6)

1. The use of a derivative of N-phenyl-2-pyrimidinamine of the formula I

where R ₁ means pyridyl attached at the carbon atom, R ₂ , R ₃ , R ₅ , R ₆ and R _{8 are} hydrogen, R ₄ means lower alkyl, R ₇ means a radical of formula II

where R ₉ means hydrogen, X means oxo, n is 0 and R ₁₀ means 4-methylpiperazinylmethyl, or a salt of such a compound containing at least one salt-forming group for the manufacture of a medicament for the treatment of mastocytosis. 2. The use of the compounds of formula I according to claim 1, which is N- {5- [4- (4-methylpiperazinomethyl) benzoylamido] -2-methylphenyl} -4- (3-pyridyl) -2-pyrimidinamine, or its pharmaceutically acceptable salt. 3. The use of the compounds of formula I according to claim 1, where the compound is used in the form of its monomesylate salt. 4. A method of treating warm-blooded mammals suffering from mastocytosis, comprising administering to the mammal a compound of formula I, as defined in one of claims 1 to 3, in an amount that is therapeutically effective in the treatment of mastocytosis, where the compound of formula I may also be present in the form of a pharmaceutically acceptable salt . 5. The method according to claim 4, where the daily dose of 30-750 mg of 4- (4-methylpiperazin-1-ylmethyl) -N- [4-methyl-3- (4-pyridin-3-yl) pyrimidin-2-ylamino ) phenyl] benzamide or a pharmaceutically acceptable salt thereof is administered to a human. 6. The method according to claim 5, where the daily dose is 30-600 mg of 4- (4-methylpiperazin-1-ylmethyl) -N- [4-methyl-3- (4-pyridin-3-yl) pyrimidin-2- ilamino) phenyl] benzamide or a pharmaceutically acceptable salt thereof.