Novel compounds, use and production thereof

FIELD: chemistry.

SUBSTANCE: invention relates to a compound, which is N3-1H-indol-5-yl-5-pyridin-4-ylpyrazine-2,3-diamine, or a pharmaceutically acceptable salt thereof, which can act as inhibitors of protein kinase, especially FLT3 tyrosine kinase. The invention also relates to a pharmaceutical composition which contains said compound in combination with another molecularly directed (target) agent, which is a traditional cytotoxic agent or a compound used after chemotherapy, supporting therapy targeted on stem cells and in case of MLL rearrangement acute lymphoblastic leukaemia in children.

EFFECT: obtaining a novel compound which can be used in medicine for preventing or treating haematological malignant growths such as AML, MLL, T-ALL, B-ALL and CMML, myeloproliferative diseases, autoimmune diseases and skin diseases, such as psoriasis and atopic dermatitis.

16 cl, 2 tbl, 26 ex

The technical field

The present invention relates to compounds of pyrazine, which act as inhibitors of protein kinases, particularly of the type FLT3-tyrosine kinase. The invention also relates to pharmaceutical means, comprising these compounds and to the use of compounds for the manufacture of a medicinal product for the treatment of hematological malignancies such as AML, MLL T-ALL, B-ALL and CMML, myeloproliferative diseases and other proliferative diseases such as cancer, autoimmune diseases, and skin diseases such as psoriasis and atopic dermatitis.

The level of technology

Protein kinase involved in regulation of cellular metabolism, proliferation, differentiation and viability. Protein kinases phosphorylate proteins on serine/treningowy or tyrosinosis residues. Activation of one class of kinases usually leads to the activation of more than one signal path through the signal cross-action. The receptors tyrosinekinase (RTK) are the main type of receptors on the cell surface, in which the intracellular part of the receptor has a kinase domain. Activating ligands represent a peptide/protein hormones, such as FL-ligand, vascular endothelial growth factor (VEGF), epidermal restuaurants (EGF), fibroblastic growth factor (FGF), nerve growth factor (NGF), platelet-produced growth factor (PDGF), insulin and the like, the Binding of ligand to the extracellular domain of the RTK results in dimerization of the receptor and conformational change that activates the kinase site of the intracellular domain. Kinase activity leads to a cascade of signal transduction through phosphorylation of other proteins that regulate cellular physiology and systems of gene expression (as an overview, see Schlessinger, J. (2000) Cell 103: 211-225; and Blume-Jensen P. & Hunter T. (2001) Nature 411: 355-365). Intracellular signaling proteins activated in the signaling cascade may be other kinases and/or proteins involved in transcription and translation. There are several families of intracellular kinases. The Janus kinase (JAK) family of tyrosine kinases (JAK l, 2, 3, and Thy 1) is activated by interaction with other proteins (see O'shea, J.J. et al. (2002) Cell 109 (Suppl.) 121-131 and are there links). Serine/threonine kinases, such as proteinkinase C (PKC) family of isozymes and mitogen-activated kinase (MAP-kinase family), are also involved in regulation of cell viability, proliferation and differentiation. PKC-isoenzymes are activated by calcium and diacylglycerol is an allosteric activator of some member is in the family of PKC (alpha, beta, gamma). Intracellular kinases interact with other proteins and often move to other compartments when activated (see Manning, G. et al. (2002) Science 298: 1912 - 1934; Martin. P.M. & Hussaini L.M. (2005) Expert Opin. Ther. Targets 9(2) 299-313 and are there links). Membrane Association may be governed by monitorowania, as in the case of PKC isoenzymes. Nuclear Association described for several different classes of kinases. MAP-kinase activated by other proteins and are able to move into the nucleus, where proteins involved in transcription and cell cycle regulation and differentiation are phosphorylated.

In the process of normal development and differentiation as activation and deactivation of the kinase strictly regulated. Oncogenic mutations leading to constitutive active kinase, can transform normal cells into malignant tumor cells. Activated mutation may thought a chromosomal translocation that is conductive to increased protein production of a merger, for example, as in the case of chronic lymphocytic leukemia in which ABL-tyrosinekinase domain is combined with the BCR protein (as an overview, see Ostman, A. (2007) Helix Review Series Oncology 2: 2-9; and Deininger, M. et al. (2005) Blood 105: 2640-2653).

Under normal hematopoesis, FLT3 active under myeloblast, but FLT3 activity then oppressed when normally the hematopoietic differentiation into Mature blood cells (Gilliand, D.G, & Griffin, J.D. (2002) Blood 100: 1532-1542; Weisel, K.C. et al. (2007) Ann. N.Y. Acad. Sci. 1106: 190-196). In acute myelogenous leukemia (AML) expression of FLT3 high in most patients (70-90%) (Carow, C.E. et al. (1996) Blood 87 (3): 1089-1096; and Rosnet, O. et al. (1993) Crit. Rev. Oncogenesis 4: 595-613). In addition, FLT3 kinase activity is elevated in one third of patients because of internal tandem duplications in ikolomani position (FLT3-ITD), leading to dimerization of the ligand-independent receptor and constitutively active kinase. FLT3-ITD is a foreshadowing of symptomatic marker with a statistically significant decrease in survival was examined group of patients with the mutation, especially in the case when the affected both alleles. There are also activating point mutations (FLT3-PM) for FLT3 described in AML patients. These activating mutations can be detected in the activation loop of the kinase domain (AL-mutation) or kolomanbrunnen domain (JM-mutations). As an overview, see Carow, C.E. et al. (1996) Blood 87 (3): 1089-1096; Tickenbrock, L. et al. (2006) Expert Opin. Emerging Drugs 11(1): 153 to 165; Anjali S. & Advani, A.S. (2005) Current Pharmaceutical Design 11: 3449-3457; B.H. Lee et al. (2007) Cancer Cell 12: 367-380); Stam, R.W. et al. (2005) Blood 106(7): 2484-2490; and are there links. In addition, FLT3-ITD or FLT3-PM was detected in the subgroup of patients with other lymphoid or myeloid malignant tumors, such as MLL T-ALL and CMML, and high FLT3 activity was described in the case of B-ALL (as a researcher is as a review see Lee, B.H., et al. (2007) Cancer Cell 12: 367-380.

However, FLT3 activity is an integral part of the normal hematopoesis. In that case, when the proliferation of immature blast cells in the bone marrow unregulated by overstimulation kinases such as FLT3, as a result you can get a reduction in the number of other hematopoietic cells. Blast cells then enter the bloodstream instead of Mature differentiated cells. The state of acute leukemia results in anemia and neutropenia. Thus, blocking harmful kinase activity can reduce the proliferation of blast cells and reduce the risk of leukemic state. Various inhibitors of FLT3 kinase tested on models of AML and clinical symptoms involving FLT3 (Cheng, Y. & Paz, K. (2008) I Drugs 11(1): 46-56; Kiyoi, H. et al. (2007) Clin. Cancer Res. 13(15): 4575-4582; Roboz, G.J. et al. (2006) Leukemia 20: 952-957; Tse, K-F. et al. (2002) Leukemia 16: 2027-2036; Smith, B.D. et al. (2004) Blood 103: 3669-3676; Knapper, S. et al. (2006) Blood 108 (10): 3494-3503; and Furukawa, Y. et al. (2007) Leukemia 21: 1005-1014). AML cell line MV4-11 carries FLT3-ITD. This cell line is very sensitive in testing the viability/proliferation of FLT3 inhibitors activity. However, in the case of ex-vivo cells of the patient is also a clash between signalling pathways, molecules, activated after FLT3 receptor can also be activated by other kinases. Knapper et al. 2006 showed that d is the same if the FLT3 autophosphorylation was suppressed in cells of a patient after exposure to inhibitors of FLT3, the level of phosphorylation after effectors suppress STAT and ERK was not reduced, possibly due to dysregulation of other signaling pathways, irrespective of FLT3-phosphorylation.

The activity of FLT3 and other RTK is regulated by autophosphorylation and compensation process, the phosphorylation of the receptor is then eliminated by specific phosphatases, which are also subject to regulation. The dysregulation of the compensation process and dephosphorylation involving phosphatases may also have an effect on RTK activity and, thus, to make changes in viability and cell proliferation. Because there are several areas of regulation, the kinase inhibitor is necessary to have some form of regarding the selectivity of interaction with the target and mechanism of action for the effective inhibition of proliferation and viability of malignant tumors or proliferative diseases.

Disclosure of inventions

This invention relates in General to certain compounds pyrazine that can act as inhibitors of the receptor tyrosine kinase FLT3, and relates to pharmaceutical compositions and methods.

Despite the fact that it is not vested with the hope related with theory, believed that the compounds described in this document can be used, for example, to treat or prevent the treatment of hematological malignancies, such as acute myelogenous leukemia (AML); undifferentiated leukemia (MLL); T-cell type acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL); chronic myelomonocytic leukemia (CMML); myeloproliferative diseases; other proliferative diseases such as cancer; autoimmune diseases; and diseases of the skin, such as psoriasis and atopic dermatitis.

The compounds also can be used in combination with molecular targeted agents, such as traditional cytotoxic agent or compound that is used in postchemotherapy, therapy, focused on the preservation of stem cells, and in MLL-rearrangement pediatric acute lymphoblastic leukemia.

In the first aspect of the invention provides a compound of formula (I) and geometrical isomers, racemates, tautomers and optical isomers him, and pharmaceutically acceptable salts, hydrates, N-oxides and physiologically hydrolyzable and acceptable esters and any form of their prodrugs:

where

R¹selected from the group consisting of

(a) indelicately,

(b) cyclohexyl,

(c) hydroxycyclohexyl,

(d) 1,3-benzothiazolyl,

(e) C_1-3-alkyl-1,3-benzothiazolyl,

(f) benzothieno,

(g) indolyl,

(h) ndazole,

(i) C_1-3-alcyonaria,

(j) carboxyaniline,

(k) C_1-3-alkoxycarbonylmethyl,

(l) carbamoylethyl,

(m) 4-methylpiperazin-1-ylcarbonyl,

(n) carboxymethylthio,

(o) acetylaminophenol and

(p) C_1-3-alkylbenzoates;

R²selected from the group consisting of

(a) pyridinyl,

(b) torpedine,

(c) chloropyridinyl,

(d) C_1-3-alkoxyphenyl,

(e) tanila,

(f) furil,

(g) phenyl,

(h) ftoheia,

(i) hydroxyphenyl,

(j) cyanophenyl,

(k) hydroxymethylene,

(l) AMINOPHENYL,

(m) carbamoylmethyl,

(n) a C_1-3-alkylaminocarbonyl,

(o) dimethylaminocarbonylmethyl,

(p) (C_1-2-alkoxy-C_2-3-alkylaminocarbonyl)phenyl,

(q) (cyano-C_2-3-alkylaminocarbonyl)phenyl,

(r) (dimethylamino-C_2-3-alkylaminocarbonyl)phenyl,

(s) - N-methoxy-N-metilaminopropionitrila,

(t) morpholine-4-ylcarbonyl,

(u) piperidine-1-ylcarbonyl and

(v) hineline;

R³represents hydrogen or NH₂;

provided that the compound is not a

4-(6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)benzamide;

N'-(1H-indol-5-yl)-5-(quinoline-5-yl)pyrazin-2,3-diamine;

5-(3-AMINOPHENYL)-N'-(1H-indol-5-yl)pyrazin-2,3-diamine;

3-[5-amino-6-(1H-indol-5-ylamino)pyrazinyl]phenol;

4-[5-amino-6-(1H-ind the l-5-ylamino)pyrazinyl]phenol; or

1-methyl-N-[6-(2-pyridinyl)pyrazinyl]-1H-benzimidazole-2-amine.

A preferred group of compounds of the invention are the compounds of formula (I), where R is a H, forming the compounds of formula (Ia):

where

R¹selected from the group consisting of

(a) hydroxycyclohexyl,

(b) C_1-3-alkyl-1,3-benzothiazol-5-yl,

(c) 1,3-benzothiazolyl,

(d) benzothieno,

(e) indolyl,

(f) C_1-3-alcheringa-5-Il,

(g) carboxyaniline,

(h) C_1-3-alkoxycarbonylmethyl and

R²selected from the group consisting of

(a) pyridinyl,

(b) torpedine and

(c) carbamoylmethyl.

A more preferred group of compounds of formula (Ia) are those in which R¹selected from the group consisting of

(a) 4-hydroxycyclohexyl,

(b) 2-methyl-1,3-benzothiazol-5-yl,

(c) 1,3-benzothiazol-5-yl,

(d) indol-5-yl,

(e) indol-6-yl, and

R²selected from the group consisting of

(a) 4-pyridinyl,

(b) 2-fluoro-4-pyridinyl and

(c) 4-carbamoylmethyl.

Preferred compounds of formula (Ia) are

N-(6-pyridin-4-Alperin-2-yl)-1H-indol-5-amine,

N-[6-(2-herperidin-4-yl)pyrazin-2-yl]-1H-indol-5-amine,

N-(6-pyridin-4-Alperin-2-yl)-1H-indol-6-amine,

N-(6-pyridin-4-Alperin-2-yl)-1,3-benzothiazol-5-amine,

2-methyl-N-(6-pyridin-4-Alperin-yl)-1,3-benzothiazol-5-amine,

4-[6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide and

4-{6-[(4-hydroxycyclohexyl)amino]pyrazin-2-yl}benzamide.

A preferred group of compounds of the invention are the compounds of formula (I), where R³represents NH₂forming the compounds of formula (Ib)

where

R¹selected from the group consisting of

(a) indolamine,

(b) cyclohexyl,

(c) hydroxycyclohexyl,

(d) C_1-3-alkyl-1,3-benzothiazolyl,

(e) benzothieno,

(f) indolyl,

(g) indazole,

(h) C_1-3-alcheringa-5-yl and

(i) carbamoylethyl;

R²selected from the group consisting of

(a) pyridinyl,

(b) chloropyridinyl,

(c) torpedine,

(d) C_1-3-alkoxyphenyl,

(e) tanila,

(f) furil,

(g) phenyl,

(h) ftoheia,

(i) hydroxyphenyl,

(j) cyanophenyl,

(k) hydroxymethylene,

(l) AMINOPHENYL,

(m) carbamoylmethyl,

(n) a C_1-3-alkylaminocarbonyl,

(o) dimethylaminocarbonylmethyl,

(p) (C_1-2-alkoxy-C_2-3-alkylaminocarbonyl)phenyl,

(q) cyano-C_2-3-alkylaminocarbonyl)phenyl,

(r) (dimethylamino-C_2-3-alkylaminocarbonyl)phenyl,

(s) (N-methoxy-N-metilaminopropionitrila,

(t) (piperidine-1-ylcarbonyl)phenyl,

(u) (morpholine-4-ylcarbonyl)phenyl,

(v) chinoline.

More prepost is consistent group of compounds of Formula (Ib) is such that in which R¹selected from the group consisting of

(a) 2-(indol-3-yl)ethyl,

(b) 4-hydroxycyclohexyl,

(c) indol-5-yl,

(d) indol-4-yl,

(e) indazol-5-Il,

(f) 2-methylindol-5-yl, and

R²selected from the group consisting of

(a) 3-pyridinyl,

(b) 4-pyridinyl,

(c) 2-chloropyridin-4-Il,

(d) 3-tanila,

(e) 3-furil,

(f) 3-ftoheia,

(g) 3-hydroxyphenyl,

(h) 4-cyanophenyl,

(i) 4-AMINOPHENYL,

(j) 4-carbamoylmethyl,

(k) 3-carbamoylmethyl,

(l) 4-dimethylaminocarbonylmethyl,

(m) 4-[(2-methoxyethyl)aminocarbonyl]phenyl,

Preferred compounds of formula (Ib) are

N3-1H-indol-5-yl-5-pyridin-4-Alperin-2,3-diamine,

N3-1H-indol-5-yl-5-pyridin-3-Alperin-2,3-diamine,

5-(2-chloropyridin-4-yl)-N3-1H-indol-5-Alperin-2,3-diamine,

N3-(2-methyl-1H-indol-5-yl)-5-pyridin-4-Alperin-2,3-diamine,

N3-(2-methyl-1H-indol-5-yl)-5-pyridin-3-Alperin-2,3-diamine,

N3-1H-indol-4-yl-5-pyridin-4-Alperin-2,3-diamine,

N3-1H-indol-5-yl-5-(3-thienyl)pyrazin-2,3-diamine,

5-(3-furyl)-N3-1H-indol-5-Alperin-2,3-diamine,

N3-1H-indol-5-yl-5-phenylpyridine-2,3-diamine,

5-(3-forfinal)-N3-1H-indol-5-Alperin-2,3-diamine,

3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide,

4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide,

4-{5-amino-6-[(2-methyl-1H-indol-5-yl)amino]pyrazin-2-yl}benzamide,

4-[5-amino-6-(1H-indol-5-ylamino)feast of the Jn-2-yl]-N-(2-methoxyethyl)benzamide,

4-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-cyanoethyl)benzamide,

4-[5-amino-6-(1H-indol-4-ylamino)pyrazin-2-yl]benzamide,

N3-[2-(1H-indol-3-yl)ethyl]-5-pyridine-4-Alperin-2,3-diamine,

N3-[2-(1H-indol-3-yl)ethyl]-5-pyridin-3-Alperin-2,3-diamine,

4-(5-amino-6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)benzamid,

4-(5-amino-6-{[2-(1H-indol-3-yl)ethyl]amino}pyrazin-2-yl)-N,N-dimethylbenzamide,

5-(4-AMINOPHENYL)-N3-[2-(1H-indol-3-yl)ethyl]pyrazin-2,3-diamine,

TRANS-4-[(3-amino-6-pyridin-4-Alperin-2-yl)amino]cyclohexanol,

3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]phenol,

N3-1H-indazol-5-yl-5-pyridin-4-Alperin-2,3-diamine,

4-[5-amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)benzamide and

4-[5-amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]benzamide.

In one aspect the present invention relates to a compound of formula I for use in therapy, particularly for use in the treatment or prevention associated with FLT3 diseases. Examples associated with FLT3 diseases include acute myelogenous leukemia (AML); undifferentiated leukemia (MLL); T-cell type acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL); chronic myelomonocytic leukemia (CMML). The present invention also relates to the compound of formula (I) for use in the treatment or prevention of hematological diseases, connected the unregulated kinase activity such as myeloproliferative diseases; other proliferative diseases such as cancer; autoimmune diseases; and diseases of the skin, such as psoriasis and atopic dermatitis.

In another aspect, the present invention relates to a pharmaceutical composition comprising a compound of formula (I) as active ingredient, in combination with a pharmaceutically acceptable diluent or carrier, especially for use in the treatment or prevention associated with FLT3 disease.

In one aspect the present invention relates to a method of treatment of a patient, human or animal suffering from associated with FLT3 disease. In the following aspect the present invention relates to a method of treatment of a patient, human or animal suffering from hematological malignancies such as acute myelogenous leukemia (AML); undifferentiated leukemia (MLL); T-cell type acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL); chronic myelomonocytic leukemia (CMML), and other hematological diseases such as myeloproliferative diseases; other proliferative diseases such as cancer; autoimmune diseases; skin diseases such as psoriasis and atop the ical dermatitis. The method can include the introduction of a patient (e.g., human or animal, dog, cat, horse, cow)in need, an effective amount of one or more compounds of the formula (I), their salts or compositions containing the compounds or salts.

The methods described in this document include those in which the patient is seen as in need of individually assigned treatment. The definition of the patient as in need of such treatment can be based on an assessment of the patient or the specialist in the field of health and can be subjective (e.g., point of view) or objective (e.g., defined by a test or diagnostic method).

In other aspects the invention relates to a method of treatment of a patient suffering from associated with FLT3 diseases or disorders, or are associated with FLT3 disease or violation, including the introduction of the specified patient in need, an effective amount of the compounds of formula I or pharmaceutical composition, so that the specified patient being treated for the aforementioned diseases or disorders.

In the following aspect the present invention relates to the use of compounds of formula (I) (for example, as a drug) for the treatment of diseases, disorders or pathological conditions associated with nagulat is through the activity of FLT3 kinase, as described in this document.

In another aspect, the present invention relates to the use of compounds of formula (I) in the manufacture of a medicinal product containing a compound of formula I for the treatment of diseases, disorders or pathological conditions associated with undesirable activity of FLT3 kinase, as described in this document.

One aspect of the present invention relates to pharmaceutical compositions comprising an effective amount of a combination of an inhibitor of the receptor tyrosine kinase FLT3, in accordance with formula (I), and other molecular targeted agent, preferably, traditional cytotoxic agent or compound that is used in postchemotherapy, therapy, focused on the preservation of stem cells, and in MLL-rearrangement pediatric acute lymphoblastic leukemia; and, optionally, a pharmaceutically acceptable carrier.

Another aspect of the invention provides a method of prevention or treatment of hematological malignancies, myeloproliferative disorders, and other proliferative disorders, autoimmune disorders and skin diseases, comprising the administration to a patient, human or animal in need of this, an inhibitor of the receptor tyrosine kinase FLT3, in accordance with formula (I)simultaneously or sequentially with other molecular e.g. is run by the agent, preferably, with traditional cytotoxic agent, or a compound used in postchemotherapy, therapy, focused on the preservation of stem cells, and in MLL-rearrangement pediatric acute lymphoblastic leukemia; in quantities sufficient to provide a therapeutic effect.

And yet another aspect of the invention relates to an inhibitor of the receptor tyrosine kinase FLT3, in accordance with formula (I), together with other molecular targeted agent, such as traditional cytotoxic agent or compound that is used in postchemotherapy, therapy, focused on the preservation of stem cells, and in MLL-rearrangement pediatric acute lymphoblastic leukemia; for the manufacture of a medicinal product for the treatment of hematological malignancies, myeloproliferative disorders, and other proliferative diseases, autoimmune disorders and skin diseases.

Another aspect of the invention relates to a method for producing a pharmaceutical composition, in which the inhibitor of the receptor tyrosine kinase FLT3, in accordance with formula (I), and other molecular targeted agent, such as traditional cytotoxic agent or compound that is used in postchemotherapy, therapy, focused on the preservation of stem cells, and in MLL-rearrangement pediatric acute is th lymphoblastic leukemia; in total therapeutic quantity, mix until smooth with a pharmaceutically acceptable carrier.

And yet, in another aspect, the invention relates to a product containing an inhibitor of the receptor tyrosine kinase FLT3, in accordance with formula (I), optionally including other molecular targeted agent, such as traditional cytotoxic agent or compound that is used in postchemotherapy, therapy, focused on the preservation of stem cells, and in MLL-rearrangement pediatric acute lymphoblastic leukemia; as a combined medicinal product for simultaneous, separate or sequential use in the treatment of hematological malignancies, myeloproliferative disorders, and other proliferative diseases, autoimmune disorders and skin diseases.

Another aspect of the present invention relates to a method for obtaining compounds in accordance with formula (I) of the invention, including the interaction of 2-amino-3,5-dibromopyrazine with the corresponding amine, followed by reaction mix Suzuki. Namely, the method of obtaining the compounds in accordance with formula (I) of the invention, including one or more of the following stages: 2-amino-3,5-dibromopyrazine (3 EQ.) and the appropriate amine was dissolved in 4 ml of water and the resulting inthe mixture is heated to 195°C for 1 hour. Add water and ethyl acetate and the phases are separated. The aqueous phase is extracted again with ethyl acetate. The combined organic phase washed (water or brine) and concentrated, obtaining the crude mixture of product and unreacted amine or alcohol. The crude mixture was used without further purification or characterization in a subsequent Suzuki reaction, which is carried out in accordance with standard protocols Suzuki, published in the literature.

The chemical reagents used in the synthetic methods described in this document may include, for example, solvents, reagents, catalysts, reagents introducing protective groups and the removal of the protective groups. The methods described above may also additionally include stage either before or after the stages described specifically in this document, to add or remove suitable protective groups for the purpose, primarily, to ensure the synthesis of compounds. In addition, various synthetic stage can be performed in an alternate sequence or in a different order to obtain the target compounds. Synthetic chemical transformation processes applicable for the synthesis of related compounds, are known in the art and include, for example, those described in publications R. Larock, Comprehensive Orgnic Transformations, VCH Publishers (1989); L. Fieser, M. Fieser, Fieser and Fieser''s Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and later editions.

Ways of implementation of the reactions described above are well known to the person skilled in the art. The necessary starting materials for preparing compounds of formula (I) are either known or can be obtained by analogy with getting known compounds. The compounds of formula (I) may contain one or more chiral carbon atoms, and consequently, they can be obtained in the form of optical isomers, e.g. in the form of a pure enantiomer or as a mixture of enantiomers (racemate), or as a mixture containing diastereomers. Separation of mixtures of optical isomers of obtaining pure enantiomers is well known in the art and, for example, can be achieved by fractional crystallization of salts with optically active (chiral) acids or by chromatographic separation on chiral columns. All isomeric forms possible (pure enantiomers, diastereomers, tautomers, racemic mixtures and unequal mixtures of two enantiomers) described for compounds that fall under the scope of the invention. In the case where the connection described in this document contain olefinic double bond with geom the electrical asymmetry, suggest that they include both TRANS and CIS (E and Z geometric isomers.

The compounds of formula (I) can be used by themselves or can be allocated in the form of their pharmacologically acceptable salts (salts obtained by the joining of acid or base). Under pharmacologically acceptable additive salts mentioned above imply such, which include therapeutically active, non-toxic, formed by the joining of the acid or base form salts, which compounds are able to form. Compounds that have the basic properties may be converted into their pharmaceutically acceptable acid additive salt by processing the basic form of the corresponding acid. Typical acids include inorganic acids such as hydrochloric, Hydrobromic, iodomethane, sulfuric acid, phosphoric acid; and organic acids such as formic acid, acetic acid, propanoic acid, hydroxyestra acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzosulfimide, toluensulfonate, methanesulfonate, triperoxonane acid, fumaric acid, succinic acid, malic acid, grape acid, citric acid, salicylic acid, para-and insulicola acid, AMOVA acid, benzoic acid, ascorbic acid and the like. Typical primary additive salt forms are sodium, potassium, calcium salts, and salts with pharmaceutically acceptable amines such as ammonia, alkylamines followed, benzathine, and amino acids, such as arginine and lysine. The term "additive salt", as used in this document also includes a solvate, which compounds and their salts are capable of forming, such as, for example, hydrates, alcoholate and the like.

For clinical use of the compounds of the invention are included in the formulation of pharmaceutical compositions for oral, rectal, parenteral or other techniques. Pharmaceutical compositions are typically prepared by mixing the active substance or its pharmaceutically acceptable salt with standard pharmaceutical excipients. Examples of excipients are water, gelatin, Arabic gum, lactose, microcrystalline cellulose, starch, sodium salt glycolate, starch, calcium phosphate, magnesium stearate, talc, colloidal silicon dioxide and the like. Such formulation can also contain other pharmacologically active agents and standard excipients, such as stabilizers, moistening agents, emulsifiers, in parametersa substances, the buffer components and the like. Typically, the number of active connections is in the range of 0.1 to 95% by weight of the medicinal product, preferably in the range of 0.2-20% of the mass. in medicinal products for parenteral use, and more preferably in the range of 1-50 wt%. in medicines for perorating introduction.

The dosage form can then be obtained by known methods, such as granulation, extrusion, microencapsulation, coating covering the shell by spraying method and the like Dosage forms can be obtained by standard methods in the form of a dosed drug directory forms of tablets, capsules, granules, powders, syrups, suspensions, suppositories or injectable forms. Liquid dosage forms may be obtained by dissolution or suspension of the active substance in water or in other suitable environments. Tablets and granules may be coated standard of production.

The levels of dose and frequency of intake dosage of a particular compound vary depending on various factors, including the specific activity of the applied compound, the metabolic stability and length of action of that compound, the age of the patient, body weight, General health, sex, diet, mode is the introduction the rate of excretion, combination with other drugs, the severity of a subject to treatment of a pathological condition, the tolerability of the patient. The dose may, for example, range from about 0.001 mg to about 100 mg per kg of body weight, administered once or multiple doses, for example, from about 0.01 mg to about 1000 mg each. Usually this dose orally, but parenteral administration can be selected.

Definition

The following definitions should be used throughout the description of the invention and are given at the end of the claims.

The terms "associated with FLT3 illness" and "disease or pathological condition associated with undesired activity of FLT3" are used interchangeably in this document to refer to any breach or symptom involving FLT3 in the process of presentation of the disease or symptom. Associated with FLT3 disease thus include, for example, but not limited to, hematological malignancies such as acute myelogenous leukemia (AML); undifferentiated leukemia (MLL); T-cell type acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL) and chronic myelomonocytic anemia (CMML).

Unless otherwise specified, the term "C_1-6-alkyl" denotes an alkyl group with unbranched or branched chain, having from 1 to 6 carbon atoms. Examples of the specified C_1-6the alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and pentyl and hexyl from unbranched and branched chain. As part of the range of C_1-6-alkyl" all of its sub-groups are considered, since C_1-5-alkyl, C_1-4-alkyl, C_1-3-alkyl, C_1-2-alkyl, C_2-6-alkyl, C_2-5-alkyl, C_2-4-alkyl, C_2-3-alkyl, C_3-6-alkyl, C_4-5-alkyl, etc. Similarly, "aryl-C_1-6-alkyl" denotes a C_1-6is an alkyl group, substituted aryl group. Examples include benzyl, 2-phenylethyl, 1-phenylethyl and 1-naphthylmethyl.

Unless otherwise specified, the term "C_1-3-alkoxy" denotes alkoxygroup with unbranched or branched chain, having from 1 to 3 carbon atoms. Examples of these C_1-3-alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy. As part of the range of C_1-3-alkoxy" all of its sub-groups are considered, since C_1-2-alkoxy and C_2-3-alkoxy.

Unless otherwise specified, the term "C_1-3-alkoxycarbonyl" means alkoxygroup with unbranched or branched chain, having from 1 to 3 atoms angle of the ode, associated with the carbonyl group. Examples of these C_1-3-alkoxycarbonyl include methoxycarbonyl, etoxycarbonyl, isopropoxycarbonyl. As part of the range of C_1-3-alkoxycarbonyl" all of its sub-groups are considered, since C_1-2-alkoxycarbonyl and C_2-3-alkoxycarbonyl.

"Pharmaceutically acceptable" means suitable in obtaining pharmaceutical compositions, which are completely harmless, non-toxic and biologically as well as otherwise may not be unacceptable, and includes applicable for veterinary use as well as for people for pharmaceutical use.

"Treatment", as used in this document includes the prevention of the above diseases or pathological conditions, or improvement or elimination of the disease, once established.

"Effective amount" means an amount of compound that provides a therapeutic effect to the subject being treated. therapeutic effect may be objective (for example, measured by some test or marker) or subjective (e.g., the patient appears signs of therapeutic effect or he feels an effect).

The term "Proletarskoye form" means a pharmacologically acceptable derivative such as ester or amide, such is proizvodnje, which is biotransforming in the body with the formation of the active drug. Reference is made to Goodman and Gilman^'s, The Pharmacological basis of Therapeutics, 8^thed., Mc-Graw-Hill, Int. Ed. 1992, "Biotransformation of Drugs", p. 13-15; and "The Organic Chemistry of Drug Design and Drug Action" by Richard B. Silverman. Chapter 8, p. 352. (Academic Press, Inc. 1992. ISBN 0-12-643730-0).

Combinations of substituents and variables provided by this invention are only those that result in the formation of stable compounds. The term "stable"as it is used in this document, refers to compounds which are stable enough for industrial production, and to ensure the integrity of the connection during the relevant time period for application for the purposes described in this document (e.g., therapeutic administration to a patient for treatment associated with FLT3 disorders or diseases (including those described in this document), for example, hematological malignancies such as acute myelogenous leukemia (AML); undifferentiated leukemia (MLL); T-cell type acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL), and chronic myelomonocytic leukemia (CMML)).

Summary list of chemical groups in any of the definitions of the variables of the characteristics of this document includes definitions of these variables, as any single group or a combination of these groups. Summary of the example embodiment of the invention for the variable characteristics of this document includes an example embodiment of the invention, which represents one exemplary embodiment of the invention, or in combination with any other embodiments of the invention or parts thereof.

The invention will now be further illustrates the following, not limiting examples. The specific examples below are merely illustrative and do not limit the rest of the disclosure of the invention in whatever other way. Without additional research it is obvious that the average expert in the art are able, based on the description of this document, to apply the present invention in full. All cited in this publication document so included as references in its entirety.

The patterns described in this document may contain certain-NH-, -NH₂(amino) and-OH (hydroxyl) groups in which the corresponding(e) hydrogen(s) atom(s) not present(ut) explicitly; although they must be understood as-NH-, -NH₂or-OH depending on the specific case.

Methods

¹H nuclear magnetic resonance (NMR) and³ C NMR were recorded on a Bruker Advance DPX 400 spectrometer at 400.1 MHz and to 100.6 MHz, respectively. All spectra were recorded using the residual solvent or tetramethylsilane was (TMS) as internal standard.

Low-resolution electrospray ionization mass spectra (LRESIMS) were obtained using Agilent MSD mass spectrometer or Waters ZQ mass spectrometer. High-resolution electrospray ionization mass spectra (HRESIMS) were obtained on a Agilent LC/MSD TOF, which is connected with the Agilent 1100 LC system, ion source: ESI (ionization method elektrorazpredelenie), ion polarity: gender, representation of data in the form of a graphical image, a scanning range: 100-1100 Yes, the mass spectrometer parameters: fragmentor 215, skimmer 560 In och OCT RF (octpole rods) 250 Century; featured mass 121,050873 and 922,009798 (Agilent reference Mix); LC (LC): A 15 mm ammonium acetate; B 100 MeCN; flow rate of 400 µl/min, isocratic mode. Flash chromatography was performed on silica gel Merck silica gel 60 (mesh 230-400 mesh). Microwave irradiation was carried out using a Smith Creator or Optimizer (Personal Chemistry), using a 0.5-2 ml or 2.5 ml ampoules Smith Process vials, equipped with an aluminium capsule and septa. Compounds were named automatically using the ACD/NAME 6.0 (Advanced Chemistry Development, Inc., Toronto, Canada).

The results of the analytical IHMS (LCMS) was obtained using:

A: Agilent MSD mass spectrometer; Agilent 1100 system; ACE 3 8 column (C,0 mm); water with 0.1% triperoxonane acid (TFA) and acetonitrile were used as mobile phases at a flow rate of eluent, 1 ml/min gradient time of 3.0 min (gradient 10-97% acetonitrile); or

System B: Agilent MSD mass spectrometer; Agilent 1100 system; YMC ODS-AQ column (C,0 mm); water of 0.1% triperoxonane acid (TFA) and acetonitrile were used as mobile phases at a flow rate of eluent, 1 ml/min gradient time of 3.0 min (gradient 10-97% acetonitrile); or

System C: Waters ZQ mass spectrometer; Waters 996 PDA detector (DAD 215-395 nm); ACE C8 (3 μm) column (C,0 mm) (from ACT); water 10 mm ammonium acetate (pH=7) and acetonitrile were used as mobile phases at a flow rate of eluent, 1 ml/min gradient time 3.2 min (gradient of 5-100% acetonitrile).

Preparative HPLC (HPLC) was performed on a Gilson system equipped with the following:

System D: ACE C8 5 μm (21,CH mm) column. Water with 0.1% triperoxonane acid (TFA) and acetonitrile were used as mobile phases at a flow rate of eluent 25 ml/min gradient time 6 minutes; or

System E: XTerra Prep MS C18 5 μm (C mm) column. Water 50 mm NH₄HCO₃(pH=10) and acetonitrile were used as mobile phases at a flow rate of eluent 25 ml/min gradient time of 6 min; or Xterra MS C18 5 μm (C mm) column.

Water 50 mm NH₄HCO₃(pH=10) and acetonitrile were used as mobile phases at a flow rate e is uent 40 ml/min gradient time of 8.5 min; or

System F: YMC ODS-AQ 10 μm (C mm) column. Water with 0.1% triperoxonane acid (TFA) and acetonitrile were used as mobile phases at a flow rate of eluent 45 ml/min gradient time of 8.5 minutes

The following abbreviations used

DMSO (DMSO) refers to dimethyl sulfoxide,

HPLC (HPLC) refers to high performance liquid chromatography

TFOC (TFA) denotes triperoxonane acid.

Msvr (HRMS) refers to mass spectrometry, high-resolution

EXAMPLES

Methodology A:

A General method for S_NAr 2-amino-3,5-dibromopyrazine

2-Amino-3,5-dibromopyrazine, triethylamine (3 EQ.) and the appropriate amine or alcohol (3 EQ.) was dissolved in 4 ml of water and the resulting mixture was heated to 195°C for 1 hour. Water and ethyl acetate were added and the phases were separated. The aqueous phase was extracted again with ethyl acetate. The combined organic phases were washed (water and brine) and concentrated to obtain the crude mixture of product and unreacted amine or alcohol. This crude mixture was used without further purification or characterization in a subsequent Suzuki reaction.

Methodology B:

General method for the reaction of a combination of Suzuki

A mixture of personalbrain from method A (1 EQ.), the corresponding Bronevoy acid (1 EQ.), K₂CO₃(3 equiv.) and Pddppf)Cl ₂*CH₂Cl₂(0.1 EQ.) in 4 ml of dioxane/water (4:1) was heated to 150°C for 15 minutes the Mixture was filtered through a small layer of silica gel and concentrated. The crude product was purified preparative HPLC (ACE C8 column; mobile phase: 0.1% of TFA - CH₃CN), getting mentioned in the title compound as a white solid in the form of its corresponding triperoxonane salt.

The intermediate connection 1

5-bromo-N3-1H-indol-5-Alperin-2,3-diamine

Using method A and 2-amino-3,5-dibromopyrazine (100 mg) and 5-aminoindole (200 mg), was obtained 150 mg of a mixture containing a 1:1 ratio 5-aminoindole and the target product. Mass spectrum m/z 303 [M+H]⁺, which was used without additional purification or registration of its characteristics.

EXAMPLE 1

N3-1H-Indol-5-yl-5-pyridin-4-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-N3-1H-indol-5-Alperin-2,3-diamine (20 mg) and 4-pyridylamino acid (12 mg)were 1.7 mg specified in the connection header. Mass spectrum m/z 303 [M+H]⁺.

¹H NMR (400 MHz, CD₃OD) d ppm 6,48 (d, J=3,01 Hz, 1H), 7,26-7,33 (m, 1H), 7,33-to 7.50 (m, 2H), 7,92 (d, J=1.25 Hz, 1H), of 8.37 (s, 1H), 8,45 (d, J=7.03 is Hz, 2H), 8,64 (d, J=7.03 is Hz, 2H).

EXAMPLE 2

N3-1H-Indol-5-yl-5-pyridin-3-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-N3-1H-indol-5-Alperin-2,3-dia is n (20 mg) and 3-pyridylamino acid (12 mg), received 1.3 mg specified in the connection header. HRMS calculated for C₁₇H₁₄N₆: 302,1280 received: 302,1279.

¹H NMR (400 MHz, CD₃OD): of 6.49 (d, 1H, J=4 Hz), 7,29-7,46 (m, 3H), 7,88-7,94 (m, 2H), 8,02 (s, 1H), 8,64 (d, 1H, J=8 Hz), 8,84 (d, 1H, J=8 Hz), 9,19 (s, 1H).

EXAMPLE 3

4-[5-Amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide, triptorelin

Using method B and 5-bromo-N3-1H-indol-5-Alperin-2,3-diamine (20 mg) and 4-benzamid Bronevoy acid (16 mg)were 0.9 mg specified in the connection header. HRMS calculated for C₁₉H₁₆N₆O: 344,1386 found: 344,1381.

¹H NMR (400 MHz, CD₃OD): d of 6.49 (d, 1H, J=4 Hz), 7,30 (d, 1H, J=4 Hz), 7,42-7,47 (m, 2H), 7,81 (s, 1H), 7,94 (d, 2H, J=8 Hz), 8,01-of 8.06 (m, 3H).

EXAMPLE 4

5-(2-Chloropyridin-4-yl)-N3-1H-indol-5-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-N3-1H-indol-5-Alperin-2,3-diamine (20 mg) and 2-chloropyridin-4-Voronovo acid (20 mg)were 4.0 mg specified in the connection header.

EXAMPLE 5

4-[5-Amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)benzamide, triptorelin

Using method B and 5-bromo-N3-1H-indol-5-Alperin-2,3-diamine (25 mg) and [4-[[(2-methoxyethyl)amino]carbonyl]phenyl]Bronevoy acid (27 mg)were 4.2 mg specified in the connection header. Mass spectrum m/z 403 [M+H]⁺.

EXAMPLE 6

4-[5-Amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]-N-(2-cyanoethyl)benzamide, triptorelin

Using method B and 5-bromo-N3-1H-indol-5-Alperin-2,3-diamine (25 mg) and [4-(2-cyanoethylidene)phenyl]Bronevoy acid (27 mg)were 3.2 mg specified in the connection header. Mass spectrum m/z 398 [M+H]⁺.

¹H NMR (500 MHz, DMSO-d₆) d ppm 2,78 (t, J=6,70 Hz, 2H), 3,51 (sq, J=6,09 Hz, 2H), 6.42 per (s, 1H), 7,27-7,47 (m, 3H), 7,76-8,18 (m, 6H), scored 8.38 (s, 1H), 8,63-9,11 (m, 1H), 10,98 (s, 1H).

Intermediate compound 2

5-Bromo-N3-1H-indol-4-Alperin-2,3-diamine

Using method A and 2-amino-3,5-dibromopyrazine (300 mg) and 4-aminoindole (470 mg)was obtained 700 mg of a mixture containing a 1:1 ratio 4-aminoindole and target product. Mass spectrum m/z 303 [M+H]⁺, which was used without additional purification or registration of its characteristics.

EXAMPLE 7

N3-1H-Indol-4-yl-5-pyridin-4-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-N3-1H-indol-4-Alperin-2,3-diamine (25 mg) and 4-pyridineboronic acid (15 mg), received 1.2 mg specified in the connection header. HRMS calculated for C₁₇H₁₄N₆: 302,1280 found: 302,1278.

¹H NMR (400 MHz, CD₃OD) ppm 6,41 (d, J=3 Hz, 1H), 7,19 (d, J=7 Hz, 1H), 7,21-to 7.32 (m, 2H), 7,38 (d, J=7 Hz, 1H), with 8.33 (d, J=6 Hz, 2H), 8,44 (s, 1H), to 8.57 (d, J=6 Hz, 2H).

EXAMPLE 8

4-[5-Amino-6-(1H-indol-4-ylamino)pyrazin-2-yl]Ben who amide, triptorelin

Using method B and 5-bromo-N3-1H-indol-4-Alperin-2,3-diamine (25 mg) and 4-benzamid Bronevoy acid (20 mg)were 1.1 mg specified in the connection header. HRMS calculated for C₁₉H₁₆N₆O: 344,1386 found: 344,1384.

¹H NMR (400 MHz, CD₃OD) d of 6.50 (d, J=2 Hz, 1H), 7,20 (t, J=7 Hz, 1H), 7,28 (d, J=3 Hz, 1H), 7,33 (d, J=8 Hz, 1H), 7,49 (d, J=7 Hz, 1H), 7,82-of 7.96 (m, 5H).

Intermediate compound 3

5-Bromo-N3-(2-methyl-1H-indol-5-yl)pyrazin-2,3-diamine

Using method A and 2-amino-3,5-dibromopyrazine (300 mg) and 5-amino-2-methylindol (520 mg), received 400 mg of a mixture containing a 1:1 ratio of 5-amino-2-methylindol and target product. Mass spectrum m/z 319 [M+H]⁺, which was used without additional purification or registration of its characteristics.

EXAMPLE 9

N3-(2-Methyl-1H-indol-5-yl)-5-pyridin-4-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-N3-(2-methyl-1H-indol-5-yl)pyrazin-2,3-diamine (26 mg) and 4-pyridineboronic acid (14 mg), received 3.0 mg specified in the connection header. HRMS calculated for C₁₈H₁₆N₆: 316,1436 found: 316,1437.

¹H NMR (400 MHz, CD₃OD) d ppm of 2.45 (s, 3H), 7,16-7,47 (m, 3H), 7,76 (s, 1H), 8,35 (s, 1H), 8,45 (d, J=6 Hz, 2H), 8,65 (d, J=6 Hz, 2H).

EXAMPLE 10

N3-(2-Methyl-1H-indol-5-yl)-5-pyridin-3-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-3-(2-methyl-1H-indol-5-yl)pyrazin-2,3-diamine (26 mg) and 3-pyridineboronic acid (14 mg), received 3.4 mg specified in the connection header. HRMS calculated for C₁₈H₁₆N₆: 316,1436 found: 316,1434.

EXAMPLE 11

4-{5-Amino-6-[(2-methyl-1H-indol-5-yl)amino]pyrazin-2-yl}benzamide, triptorelin

Using method B and 5-bromo-N3-(2-methyl-1H-indol-5-yl)pyrazin-2,3-diamine (26 mg) and 4-benzamid Bronevoy acid (19 mg)were 2.2 mg specified in the connection header. HRMS calculated for C₂₀H₁₈N₆O: 358,1542 found: 358,1542.

¹H NMR (400 MHz, CD₃OD) d of 2.46 (s, 3H), 7,15-of 7.48 (m, 3H), to 7.77 (s, 1H), of 7.90 (s, 1H), to $ 7.91-of 7.96 (m, 2H), 8,00-to 8.12 (m, 2H).

Intermediate compound 4

5-Bromo-N3-(1H-indazol-5-yl)pyrazin-2,3-diamine

Using method A and 2-amino-3,5-dibromopyrazine (300 mg) and 5-aminoindazole (470 mg)was obtained 320 mg of a mixture containing a ratio of 1:3 5-aminoindazole and target product. Mass spectrum m/z 306 [M+H]⁺, which was used without additional purification or registration of its characteristics.

EXAMPLE 12

N3-1H-Indazol-5-yl-5-pyridin-4-Alperin-2,3-diamine, triptorelin

Using method B and 5-bromo-N3-(1H-indazol-5-yl)pyrazin-2,3-diamine (15 mg) and 4-pyridylamino acid (9 mg), received 1.3 mg specified in the connection header. HRMS calculated for C₁₆H₁₃N₇: 303,1232 found: 303,1231.

EXAMPLE 13

4-[5-Amino-6-(1H-indazol-5-the laminitis)pyrazin-2-yl]benzamide, triptorelin

Using method B and 5-bromo-N3-(1H-indazol-5-yl)pyrazin-2,3-diamine (15 mg) and 4-benzamid Bronevoy acid (12 mg), received 1.5 mg specified in the connection header. HRMS calculated for C₁₈H₁₅N₇O: 345,1338 found: 345,1335.

EXAMPLE 14

4-[5-Amino-6-(1H-indazol-5-ylamino)pyrazin-2-yl]-N-(2-methoxyethyl)benzamide, triptorelin

Using method B and 5-bromo-N3-(1H-indazol-5-yl)pyrazin-2,3-diamine (15 mg) and [4-[[(2-methoxyethyl)amino]carbonyl]phenyl]Bronevoy acid (16 mg), received 2.5 mg specified in the connection header. HRMS calculated for C₂₁H₂₁N₇O₂: 403,1757 found: 403,1751.

The intermediate compound 5

5-Bromo-N3-[2-(1H-indol-3-yl)ethyl]pyrazin-2,3-diamine

Using method A: 2-Amino-3,5-dibromopyrazine (300 mg) and tryptamine (570 mg), received 600 mg of a mixture containing a 1:1 ratio of tryptamine and target product. Mass spectrum m/z 333 [M+H]⁺, which was used without additional purification or registration of its characteristics.

EXAMPLE 15

4-[6-(1H-Indol-5-ylamino)pyrazin-2-yl]benzamide, triptorelin

5-Aminoindole (100 mg), 2,6-dichloropyrazine (100 mg) and triethylamine (135 mg) were mixed in 4 ml of acetonitrile and heated to 150°C for 1 h Aqueous saturated solution of NaHCO₃and dichloromethane was added to the reaction mixture and the phases were separated. Water the second phase was extracted with dichloromethane. The combined organic phases are washed with brine and concentrated. The crude intermediate compound, 6-chloro-N-(1H-indol-5-yl)pyrazin-2-amine, (4-aminosulphonylphenyl)baronova acid (121 mg), K₂CO₃(275 mg) and Pd(tetrakis(triphenylphosphine)) (38 mg) was dissolved in 4 ml of dioxane and 1 ml of H₂O, and the reaction mixture was heated to 100°C during the night. 1M NaOH(_aq.) and dichloromethane was added to the mixture, and the phases were separated. The aqueous phase was extracted with dichloromethane. The combined organic phases are washed with brine and concentrated. The crude product was purified preparative HPLC (ACE C8 column; mobile phase: 0.1% of TFA - CH₃CN), getting mentioned in the title compound (85 mg) as a white solid in the form of its corresponding triptoreline salt. HRMS calculated for C₁₉H₁₅N₅O: 329,1277 found: 329,1279.¹H NMR (400 MHz, CD₃OD) d 7,27 (d, J=3,01 Hz, 1H), 7,31-7,37 (m, 1H), 7,40-the 7.43 (m, 1H), 7,47-7,51 (m, 1H), 7,85-to 7.93 (m, 1H), 7.95 is-8,08 (m, 3H), 8,19 compared to 8.26 (m, 2H), scored 8.38 (s, 1H).

EXAMPLE 16

5-(3-forfinal)-N~3-1H-indol-5-Alperin-2,3-diamine. Bought from BioFocus DPI: HRMS calculated for C₁₈H₁₄FN₅: 319,123324 found: 319,123684. Mass spectrum m/z 320 [M+H]⁺.

EXAMPLE 17

5-(3-furyl)-N~3-1H-indol-5-Alperin-2,3-diamine.

Bought from BioFocus DPI: HRMS calculated for C₁₆ 13N₅O: 291,112010 found: 291,112130. Mass spectrum m/z 292 [M+H]⁺.

EXAMPLE 18

3-[5-amino-6-(1H-indol-5-ylamino)pyrazin-2-yl]benzamide. Bought from BioFocus DPI: HRMS calculated for C₁₉H₁₆N₆O: 344,138559 found: 344,138509. Mass spectrum m/z 345 [M+H]⁺.

EXAMPLE 19

N~3-1H-indol-5-yl-5-(3-thienyl)pyrazin-2,3-diamine. Bought from BioFocus DPI: HRMS calculated for C₁₆H₁₃N₅S: 307,089166 found: 307,089106. Mass spectrum m/z 308 [M+H]⁺.

EXAMPLE 20

4-{6-[(TRANS-4-Hydroxycyclohexyl)amino]pyrazin-2-yl}benzamide, triptorelin

2,6-Dichloropyrazine (500 mg), TRANS-4-aminocyclohexanol (380 mg) and triethylamine (500 mg) was dissolved in 4 ml of acetonitrile/1 ml water and the reaction mixture was heated to 150°C for 15 minutes. Water and dichloromethane were added to the mixture and the phases were separated. The aqueous phase is once again was extracted with dichloromethane. The combined organic phases were washed (water and brine) and evaporated, receiving 750 mg of the intermediate 6-chloro-N-(TRANS-4-hydroxycyclohexyl)pyrazin-2-amine with a purity of 85%. Part of this substance (30 mg), potassium carbonate (55 mg), 4-benzamide Bronevoy acid (26 mg) and Pd(tetrakis(triphenylphosphine)) (5 mg) was dissolved in 4 ml of dioxane and 1 ml of H₂O, and the reaction mixture was heated to 100°C during the night. 1M NaOH(_aq.and harmatan was added to the mixture, and the phases were separated. The aqueous phase was extracted with dichloromethane. The combined organic phases are washed with brine and concentrated. The crude product was purified preparative HPLC (ACE C8 column; mobile phase: 0.1% of TFA - CH₃CN), getting mentioned in the title compound (5.0 mg) as a white solid in the form of its corresponding triptoreline salt. HRMS calculated for C₁₇H₂₀N₄O₂: 312,1586 found: 312,1585.

EXAMPLE 21

TRANS-4-[(3-Amino-6-pyridin-4-Alperin-2-yl)amino]cyclohexanol

A suspension of 2,6-dibromo-3-aminopyrazine (6,44 g, 0,0255 mol), K₂CO₃(6.9 g, 0.05 mol) and TRANS-4-aminocyclohexanol (HCl salt) (of 7.55 g, 0.05 mol) in H₂O (10.0 ml) was heated to boiling under reflux for 72 h (homogeneous solution was quickly formed and after approximately 30 h, the solid slowly precipitated. The mixture was cooled and the insoluble solid was separated and washed with water, which gave the opportunity to get 4,336 g (59%) of intermediate compounds, TRANS-4-[(3-amino-6-bromopyrazine-2-yl)amino]cyclohexanol. To a solution of the crude substance (4,336 g, 0,0151 mol), 4-pyridylamino acid (1.84 g, 0,0151 mol) tetrakis(triphenylphosphine)palladium(0) (870 mg, 0.7 mmol; 5% mol) in PhMe (200 ml) was added an aqueous solution of 2M sodium carbonate (40 ml), and ethanol (40 ml). mesh was heated to boiling under reflux overnight. The mixture was concentrated by evaporation and insoluble dark-colored solid substance was separated by filtration. This substance was then dissolved in MeOH and perform flash chromatography on silica with EtOAc-MeOH (9:1)to give pale yellow solid (2.2 g). Subsequent elution with EtOAc-MeOH (7:1) gave additional weight of a pale yellow solid (930 mg), which was quite clogged with silica gel. Both masses solids were combined and purified preparative HPLC (ACE C8 column; mobile phase: 0.1% of TFA-CH₃CN), which gave the opportunity to obtain 2.2 g specified in the header of the product. Purity according to HPLC 100%;¹H NMR (400 MHz, DMSO-d₆) d ppm 1,33-of 1.40 (m, 4H), 1,89-of 1.92 (m, 2H), 2,01-2,04 (m, 2H), 3,47-to 3.49 (m, 1H), 3,93-of 3.97 (m, 1H), 8,29 (s, 1H), to 8.41 (d, 2H, J=5.0 Hz), 8,80 (d, 2H, J=5.0 Hz). Mass spectrum (API-ES/Positive); m/z: 286 (M+H)⁺.

EXAMPLE 22

N-(6-Pyridin-4-Alperin-2-yl)-1H-indol-5-amine

A mixture of 2,6-dichloropyrazine (0,845 g, 5,67 mmol), 5-aminoindole (0.5 g, of 3.78 mmol), BINAP (0,051 g, 0,0831 mmol), tert-butoxide sodium (0.51 g, from 5.29 mmol) and palladium acetate (0,0186 g, 0,0831 mmol) in toluene (25 ml) was heated to 85°C for 22 h under nitrogen atmosphere. CH₂Cl₂was added, the reaction mixture was filtered through Celite, and the solvent was evaporated. The residue was purified column chromatography (5% methanol in CH₂Cl₂as eluent), recip what I 0,180 g (13%) of the intermediate compound N-(6-chloropyrazine-2-yl)-1H-indol-5-amine.

¹H NMR (CD₃OD) d of 7.97 (s, 1H), to 7.84 (s, 1H), to 7.77 (s, 1H), 7,42-7,39 (d, J=8,63 Hz, 1H), 7,28-7,20 (m, 2H), 6,47-6,46 (d, J=2,83 Hz, 1H). Mass spectrum (API-ES/Positive); m/z: 245 (M+H)⁺.

A mixture of N-(6-chloropyrazine-2-yl)-1H-indol-5-amine (0,030 g, 0,123 mmol), pyridine-4-Bronevoy acid (0,018 g, 0.147 mmol), sodium carbonate (0,067 g of 0.615 mmol) and tetrakis(triphenylphosphine)palladium(0) (0,007 g 0,006 mmol) in DME:water (3:2, 5 ml) was heated to boiling under reflux for 20 hours the Reaction mixture was concentrated under reduced pressure and the obtained residue was extracted with dichloromethane. The organic layer was washed with water, brine, dried over sodium sulfate and concentrated. The crude product was purified flash chromatography (5% methanol in CH₂Cl₂as eluent)to give N-(6-pyridin-4-Alperin-2-yl)-1H-indol-5-amine (0,011 g, 31%) as a yellow solid.

¹H NMR (CD₃OD) d 8,70-8,68 (d, J=6,17 Hz, 2H), of 8.47 (s, 1H), 8,15 (s, 1H), 8,14 (d, J=1.50 Hz, 2H), of 7.96 (d, J=1.70 Hz, 1H), 7,45-the 7.43 (d, J=8,64 Hz, 1H), 7,37-to 7.35 (DD, J=10,46 and 1.83 Hz, 1H), 7,29-7,28 (d, J=3,06 Hz, 1H), of 6.49-6,48 (d, J=3,02 Hz, 1H). Mass spectrum (API-ES/Positive); m/z: 288 (M+H)⁺.

EXAMPLE 23

N-[6-(2-herperidin-4-yl)pyrazin-2-yl]-1H-indol-5-amine

A mixture of N-(6-chloropyrazine-2-yl)-1H-indol-5-amine (0.05 g, 0,205 mmol), 2-herperidin-4-Bronevoy acid (0,057 g, 0.4 mmol), sodium carbonate (0,112 g 1,025 mmol) and tetrakis(triphenylphosphine)palladium(0) (0,012 g 0,01 shall mol) in DME:water (3:2, 3 ml) was heated to boiling under reflux for 20 hours the Reaction mixture was concentrated under reduced pressure and the obtained residue was extracted with dichloromethane. The organic layer was washed with water, brine, dried over sodium sulfate and concentrated. The crude product was purified flash chromatography (5% methanol in CH₂Cl₂as eluent), getting mentioned in the title compound (0.015 g, 24%) as a yellow solid.

¹H NMR (CD₃OD) d of 8.47 (s, 1H), 8,35-of 8.33 (d, J=of 5.29 Hz, 1H), 8,16 (s, 1H), 8,01-of 8.00 (d, J=5,13 Hz, 1H), 7,94 (s, 1H), 7,78 (s, 1H), 7,45-the 7.43 (d, J=8,64 Hz, 1H), 7,35-7,33 (DD, J=10,33, 1,72 Hz, 1H), 7,29 (d, J=2,95 Hz, 1H), 6,48-6,47 (d, J=2,58 Hz, 1H). Mass spectrum (API-ES/Positive); m/z: 306 (M+H)⁺.

EXAMPLE 24

N-(6-Pyridin-4-Alperin-2-yl)-1H-indol-6-amine

A mixture of 2,6-dichloropyrazine (0,150 g, 1,006 mmol), 6-aminoindole (0,200 g and 1.51 mmol), BINAP (0,0137 g, 0,02215 mmol), tert-butoxide sodium (0,136 g, 1,409 mmol) and palladium acetate (0.005 g, 0,02215 mmol) in toluene (8 ml) was heated at 85°C for 16 h under nitrogen atmosphere. CH₂Cl₂was added, the reaction mixture was filtered through Celite and the solvent was evaporated. The residue was purified column chromatography (5% methanol in CH₂Cl₂as eluent)to give 0,070 g (33%) of intermediate compound (6-chloropyrazine-2-yl)-(1H-indol-6-yl)amine.

¹H NMR (CDCl₃) d at 8.36 (users, 1H, H), 8,08 (s, 1H), 7,92 (s, 1H), of 7.64-to 7.59 (m, 2H), 7.23 percent (s, 1H), 7,01-6,98 (d, J=of 8.37 Hz, 1H), 6.87 in (s, 1H, NH), 6,56 (s, 1H). Mass spectrum (API-ES/Positive); m/z: 245 (M+H)⁺.

A mixture of (6-chloropyrazine-2-yl)-(1H-indol-6-yl)amine (0,070 g, 0,2868 mmol), pyridine-4-Bronevoy acid (0,042 g, 0,344 mmol), sodium carbonate (0,150 g of 1.43 mmol) and tetrakis(triphenylphosphine)palladium(0) (0,0165 g, 0,0143 mmol) in DME:water (3:2, 5 ml) was heated at boiling under reflux for 20 hours the Reaction mixture was concentrated under reduced pressure and the obtained residue was extracted with dichloromethane. The organic layer was washed with water, brine, dried over sodium sulfate and concentrated. The crude product was purified flash chromatography (5% methanol in CH₂Cl₂as eluent)to give N-(6-pyridin-4-Alperin-2-yl)-1H-indol-6-amine (0,030 g of 36.5%) as a yellow solid.

¹H NMR (CD₃OD) d 8,73-8,72 (d, J=of 5.68 Hz, 2H), 8,51 (s, 1H), 8,23-to 8.20 (m, 4H), 7,56-rate of 7.54 (d, J=8,46 Hz, 1H), 7,22 (d, J=2,99 Hz, 1H), 7,14 (DD, J=10,18, 1,74 Hz, 1H), 6,45 (d, J=2,75 Hz, 1H). Mass spectrum (API-ES/Positive); m/z: 288 (M+H)⁺.

EXAMPLE 25

2-Methyl-N-(6-pyridin-4-Alperin-2-yl)-1,3-benzothiazol-5-amine

A mixture of 2,6-dichloropyrazine (0,150 g, 1,006 mmol), 5-amino-2-methylbenzothiazole (0,250 g and 1.51 mmol), BINAP (0,0137 g, 0,02215 mmol), tert-butoxide sodium (0,136 g, 1,409 mmol) and palladium acetate (0.005 g, 0,02215 mmol) in toluene (8 ml) was heated at 85°C for 16 h in atmosferarosa. CH₂Cl₂was added, the reaction mixture was filtered through Celite and the solvent was evaporated. The residue was purified column chromatography (5% methanol in CH₂Cl₂as eluent)to give 0,180 g (65%) of intermediate compounds (6-chloropyrazine-2-yl)-(2-methylbenzothiazol-5-yl)amine.

¹H NMR (CDCl₃) d to 7.59-rate of 7.54 (d, J=15.6 Hz, 2H), 7,38 (s, 1H), 7,19-7,16 (d, J=8,65 Hz, 1H), 6,98-to 6.95 (d, J=to 8.41 Hz, 1H), of 6.71 (users, 1H, NH), 2,31 (s, 3H, CH₃). Mass spectrum (API-ES/Positive); m/z: 277 (M+H)⁺. A mixture of (6-chloropyrazine-2-yl)-(2-methylbenzothiazol-5-yl)amine (0.075 g, 0,271 mmol), pyridine-4-Bronevoy acid (0,040 g, 0,326 mmol), sodium carbonate (0,143 g, 1.35 mmol) and tetrakis(triphenylphosphine)palladium(0) (0,0156 g, 0,0135 mmol) in DME:water (3:2, 5 ml) was heated to boiling under reflux for 20 hours the Reaction mixture was concentrated under reduced pressure and the obtained residue was extracted with dichloromethane. The organic layer was washed with water, brine, dried over sodium sulfate and concentrated. The crude product was purified flash chromatography (5% methanol in CH₂Cl₂as eluent)to give 2-methyl-N-(6-pyridin-4-Alperin-2-yl)-1,3-benzothiazol-5-amine (0.075 g, 86,5%) as a yellow solid.

¹H NMR (CD₃OD) d 8,82 (m, 3H), 8,72 (s, 1H), 8,45-8,44 (m, 2H), 8,35 (s, 1H), 7,92-of 7.90 (d, J=8,67 Hz, 1H), to 7.61-to 7.59 (DD, J=10,63, 1,94 Hz, 1H), 2,89 (s, 3H, CH₃). Mass spectrum (API-ES/Positive); m/z: 320 (M+H)⁺.

EXAMPLE 26

N-(6-Pyridin-4-Alperin-2-yl)-1,3-benzothiazol-5-amine

A mixture of 2,6-dichloropyrazine (0,150 g, 1,006 mmol), 5-aminobenzothiazole (0,151 g, 1,006 mmol), BINAP (0,0137 g, 0,02215 mmol), tert-butoxide sodium (0,136 g, 1,409 mmol) and palladium acetate (0.005 g, 0,02215 mmol) in toluene (8 ml) was heated at 85°C for 16 h under nitrogen atmosphere. CH₂Cl₂was added, the reaction mixture was filtered through Celite and the solvent was evaporated. The residue was purified column chromatography (5% methanol in CH₂Cl₂as eluent)to give 0,140 g (53%) of intermediate compounds, benzothiazole-5-yl-(6-chloropyrazine-2-yl)amine.

¹H NMR (CDCl₃) d 10,12 (s, 1H), 9,38 (s, 1H), 8,59 (s, 1H), they were 8.22 (s, 1H), 8,11-8,08 (d, J=8,67 Hz, 1H), 8,02 (s, 1H), 7,6-EUR 7.57 (d, J=8,67 Hz, 1H). Mass spectrum (API-ES/Positive); m/z: 263 (M+H)⁺.

The mixture benzothiazol-5-yl-(6-chloropyrazine-2-yl)amine (0.06 g, 0,228 mmol), pyridine-4-Bronevoy acid (0,043 g, 0,342 mmol), sodium carbonate (0.124 g, to 1.14 mmol) and tetrakis(triphenylphosphine)palladium(0) (0,013 g, 0,0114 mmol) in DME:water (3:2, 5 ml) was heated at the boil under reflux for 22 hours, the Reaction mixture was concentrated under reduced pressure and the obtained residue was extracted with dichloromethane. The organic layer was washed with water, brine, dried over sodium sulfate and concentrated. The crude product was purified flash chromatography (5% methanol in C ₂Cl₂as eluent)to give N-(6-pyridin-4-Alperin-2-yl)-1,3-benzothiazol-5-amine (0.035 g, 50%) as a yellow solid.

¹H NMR (CD₃OD) d 9,31 (s, 1H), 8,98 (d, J=2,02 Hz, 1H), 8,75-a total of 8.74 (d, J=5,31 Hz, 2H), 8,64 (s, 1H), 8,31 (s, 1H), 8,24 is 8.22 (d, J=5,99 Hz, 2H), 8,06-of 8.04 (d, J=8,77 Hz, 1H), 7,74-7,71 (DD, J=of 10.73, a 2.01 Hz, 1H). Mass spectrum (API-ES/Positive); m/z: 306 (M+H)⁺.

Biological methods

The ability of the compounds of the invention inhibit FLT3 can be determined through the use of tests in vitro and in vivo, are known from the prior art. Some tests kinase in vitro by inhibition of FLT3 described in the literature and include the use of cloned kinase domain and assessment of phosphorylation of the substrate peptide. In addition, cell lines expressing FLT3, is used to assess the impact on the viability and proliferation in cell tests.

Test of inhibition of the enzyme

Compounds in accordance with the invention, characterized by inhibition of FLT3 them using the following methods:

Test in vitro for the FLT3 kinase

Test for inhibition of the enzyme for tyrosinekinase domain of FLT3 was performed using the method of fluorescence polarization, Immobilized Metal Ion Affinity-Based Fluorescence Polarization (IMAP) from Molecular Devices.

Summary: kinase activity was evaluated by incubation of fluorescent peptide the first substrate with a kinase domain. After completion of the kinase reaction was added to the binding buffer. After phosphorylation of the substrate fluorescent peptide acquires the ability to bind to the coated metal nanoparticles. In the case where the substrate is associated with the nanoparticle, the speed of rotation of the peptide is reduced, and therefore, the polarization of fluorescence (fluorescence polarization (fp)) becomes high. Compounds that inhibit the kinase activity of the enzyme, the result will lead to a low degree of phosphorylation of the substrate and to a low value fp signal.

Reagents

Set buffer solutions with IMAP Progressive Binding System (Molecular Devices, #R8124):

Reaction buffer: 10 mm Tris-HCl pH to 7.2 with 10 mm MgCl₂, of 0.05% NaN₃and 0.01% Tween 20. Before using DTT brought up to 1 mm DTT final concentration (ready reaction buffer).

The binding solution was prepared from a set of buffer solutions in accordance with the manufacturer's recommendations. Linking reagent was diluted 1:1500 using 40% binding buffer A and 60% binding buffer B.

Applied enzyme FLT3 was a recombinant human FLT3 from the firm Upstate (#14-500) 7,2 Ed./ml, N-end-labeled GST, amino acids 564 - end.

Applied substrate peptide: FAM-CSKtide from the company Molecular Devices (#R7269) 20 μm, 5FAM-KKKKEEIYFFFG-NH₂.

ATP is the basic solution of 10 mm

DTT basic solution of 100 mm

Thinners is soedineniya: 0,01% Tween 20+1% DMSO in the reaction buffer. Reagents were diluted in ready reaction buffer to working solutions.

Test conditions

Final concentration:

FLT3: of 0.0125 Units/ml (depending on the party)

FAM-CSKtide: 100 nm

ATP: 100 mm

Sensitive dose connection: step eleven dilutions of 1:3, the range of concentrations 25000-0,42 nm, 5000-0,085 nm, respectively 500-0,0085 nm depending on the efficiency of the connection.

Protocol

I. Conducting kinase reaction volume of 20 μl for 1 h:

Dispense pipette into 96-well plate, black ½ square:

5 µl of the diluted compounds or environment

5 μl of substrate peptide (400 nm)

5 μl of enzyme (0,05 Ed./ml) or ready reaction buffer for non-specific background values (NSB)

5 μl of ATP (400 μm)

Close the tablet and incubate at room temperature with slight agitation

II. Incubation to bind for 2 hours (minimum time):

Add 60 ál of binding solution.

Close the tablet and incubate at room temperature with slight agitation

III. Analysis of the polarization of fluorescence:

Take measurements for fluorescein using a tablet device reader (Analyst AD), the wavelength of the excitation radiation 485 and wavelength emission 530, the readout integration time of 0.1 sec (Alternatively Victor²V Wallac 485/535 nm)

The initial concentration of test with the joining was 10 mm in 100% DMSO. When the test compounds were tested at one point in 10 and 1 micromolar concentration, was diluted in the reaction buffer as described above. Compounds with inhibitory activity more than 60% inhibition at 1 micromolar concentration were further tested depending on the dose to determine the IC₅₀using eleven graded dilutions of 1:3 (normal range from 25000 nm to 0.42 nm, more effective compounds were tested from 500 nm to 0,0085 nm). The values of the IC₅₀considered by the equation (A+((B-A)/(1+((C/x)^ΛD))))in which A is equal to min, B max is, C is IC₅₀and D is equal to the tangent of the angle of the graphic curve.

Compounds in accordance with the invention, can be of value IC₅₀between 1 nm and 2 μm (for example, between 1 nm and 1 μm, between 1 nm and 500 nm, between 1 nm and 100 nm, between 1 nm and 25 nm, between 1 nm and 10 nm).

In particular, the IC₅₀N3-1H-indol-5-yl-5-pyridin-4-Alperin-2,3-diamine (AKN-028) is 6 nm.

Cell research

AML cell line MV4-11 has FLT3-internal tandem duplication. This cell line is widely used to assess the impact of inhibitors of FLT3-kinase on the viability and proliferation.

Cells were sown with a low density in 96-well plates. Added serial dilutions of compounds and cells were incubated for 72 hours. The total number of W is nesposobnyh cells was determined, using flow cytometry, at the end of treatment, and the effects of compounds considered as % inhibition compared to processed by the environment the cells.

Conditions of cell cultivation and culture

All cells were cultured under standard conditions of cell culture, at 37°C and in an atmosphere of 5% CO₂at 90% humidity.

AML cell line MV4-11 was cultured in medium with high glucose DMEM Glutamax (4500 g/l glucose)supplemented with 10% fetal bovine serum (FBS) from Invitrogen. Cells were perseval twice a week, Dorosheva to a density of approximately 2 million cells per ml volume before replanting.

Research viability and proliferation

To determine the viability of 3000-5000 cells were sown in 50 microlitres culture medium in 96-well plate. Serial dilutions of 1:3 mortar joints from 10 mm DMSO (DMSO) basic solution was carried out in serum-free culture medium, supplemented with penicillin and streptomycin. 50 microlitres solutions serial dilution was added to the cell suspension. The final concentration of the compounds was 5 micromolar to 0.8 nm, or from 500 nm to 0.08 nm, respectively. The concentration of DMSO was maintained constant, equal to 0.05%.

At the end of processing 100 microlitres reagent for determination of viability (Guava ViaCount)was added to each well, and the number of cells and their viability was determined using flow cytometry (Guava 96-well ViaCount assay). Typically, the experiments with the processed medium (0.05% DMSO) cells cell lines were performed in triplicate throughout the experiment.

percent survival was calculated in comparison with the processed medium by cells at the end of the experiment.

Value EC₅₀calculated using the equation (A+((B-A)/(1+((C/x)^ΛD))))in which A is equal to min, B max is, C is IC₅₀and D is equal to the tangent of the angle of the graphic curve.

Results

An in vitro study with a combination of FLT3 inhibitor and chemotherapy

The serial number according to the synergistic activity of the compounds of formula (I) and standard chemotherapeutic agents used in the treatment of AML was established, as described by Brown et al. (2006) Leukemia 20: 1368-1376, and the results were analyzed using the software Calcusyn Software, in accordance with the rules of the LUT and Talalay (1981) Eur. J. Biochem.

1. The Union, representing
N3-1H-indol-5-yl-5-pyridin-4-Alperin-2,3-diamine,
or its pharmaceutically acceptable salt.

2. The compound according to claim 1 for use in the treatment of FLT3-related disorders.

3. The compound according to claim 2, intended for the treatment of hematological malignancies, myeloproliferative disorders, and other proliferative disorders, autoimmune diseases and skin diseases.

4. The compound according to claim 2, intended for the prevention of hematological malignancies, myeloproliferative disorders, and other proliferative disorders, AU is ommunic diseases and skin diseases.

5. The compound according to claim 3, intended for the treatment of hematological malignancies.

6. The compound according to claim 4, intended for the prevention of hematological malignancies.

7. The compound according to claim 5 or 6, where the Hematology violation is selected from acute myelogenous leukemia (AML); undifferentiated leukemia (MLL); T-cell type acute lymphocytic leukemia (T-ALL); B-cell type acute lymphocytic leukemia (B-ALL); chronic lymphocytic leukemia (CMML).

8. The connection according to claim 7, where the Hematology violation is acute myelogenous leukemia (AML).

9. The compound according to claim 3, intended for the treatment of skin diseases.

10. The connection according to claim 9, where the skin disease is psoriasis or atopic dermatitis.

11. Pharmaceutical composition comprising an effective amount of a combination of compounds according to claim 1 and another molecular directed (targeted) agent.

12. The pharmaceutical composition according to claim 11, in which the molecular directed (targeted) agent is a traditional cytotoxic agent or compound that is used after chemotherapy, aimed at stem cell maintenance therapy with MLL-rearrangement acute lymphoblastic leukaemia in children.

13. The drug comprising the compound according to claim 1 and another directed molecular targeted agent, designed for the treatment of hematological malignancies by sequential or simultaneous administration of components.

14. The drug is indicated in paragraph 13, in which the molecular directed (targeted) agent is a traditional cytotoxic agent or compound that is used after chemotherapy, aimed at stem cell maintenance therapy with MLL-rearrangement acute lymphoblastic leukaemia in children.

15. The drug is indicated in paragraph 13, where hematologic failure is acute myelogenous leukemia (AML).