CN101489562A

CN101489562A - JAK inhibitors for treatment of myeloproliferative disorders

Info

Publication number: CN101489562A
Application number: CNA2007800276626A
Authority: CN
Inventors: 帕维尔·多布然斯基; 布鲁斯·A·鲁杰里
Original assignee: Cephalon LLC
Current assignee: Cephalon LLC
Priority date: 2006-07-21
Filing date: 2007-07-20
Publication date: 2009-07-22
Anticipated expiration: 2027-07-20
Also published as: CN101489562B; ZA200901233B

Abstract

The invention provides a method for treating myeloproliferative disorders, myelodysplastic syndromes and other diseases, in which activation of JAK2 contributes to pathology, in a mammal comprising administering to the mammal an effective amount of a fused pyrrolocarbazole derivative wherein the fused pyrrolocarbazole derivative inhibits the activity of JAK2.

Description

The JAK inhibitor that is used for the treatment of spinal cord hypertrophy disease

Technical field

The present invention relates to spinal cord hypertrophy disease (NfPDs) and the syndromic treatment of spinal cord abnormal development.Specifically, the present invention relates to use chemical compound, particularly use fused pyrrolocarbazoles compounds for treating MPDs and spinal cord abnormal development syndrome as the JAK2 inhibitor.

Background technology

Spinal cord hypertrophy disease (MPDs) is a kind of clone malignant diseases that are characterised in that one or more hematopoietic lineage excess produce, and this hematopoietic lineage has the differentiation relatively normally that causes hypercellularity bone marrow (BM).Think that MPDs appears in single multipotency CFU-GM or the stem cell, its domination BM and blood.Show that through the hemopoietic progenitor cell that derives from MPDs patient of cultivating growth sexually revises and the somatomedin dependent/non-dependent.Molecular basis for some MPDs is not clear, replace V617F up to the single amino acids that a series of reports have recently been discerned in false kinases (pseudokinase) domain at JAK2, in polycythemia vera (PV), specially send out the molecular lesion that conduct prevails in property thrombocytosis (ET) and the chronic idiopathic myelofibrosis (CIMF).This sudden change in the ET of the PV patient of 75%-97% and Yue 40%-50% and CIMF patient, be found (people such as James, 2005, Trends in Molecular Medicine 11,546-554).Importantly be, 85 kinds of other kinase whose sudden changes of in inhibition (autoinhibitory) domain or kinase domain, not finding other (people such as Levine, 2005, Cancer Cell 7,387-397).In addition, also in spinal cord abnormal development syndrome (MDS), discerned the V617F sudden change.Based on the JAK2 structure of prediction, the V617F displacement has destroyed interacting from inhibition between JH2 and proteinic kinases (JH1) domain.Therefore, the V617F mutant has to form active and be endowed the somatomedin dependent/non-dependent and form STAT5 in the BaF3/EPOR cell when expressing and activates (people such as Levine, Cancer Cell 2005,7,387-397; People such as Kralovics, 2005, N.Engl J.Med.352,1779-1790)).The CFU-E of delivery V617F sudden change grows and forms the red assembly of endogenous to fall (EEC) (sign of MPDs) when the exogenous erythropoietin of shortage (EPO), and the JAK2 inactivation of siRNA mediation reduces EEC formation.At last, with expressing the V617F mutant but not the pathological characters that the mice that the Os Mus myelocyte of wild type JAK2 is transplanted develops into nearly similar in appearance to the PV of philtrum, the height that comprises hemoglobin/hematocrit increases, leukocytosis, hyperplasia megakaryocytic, extramedullary hemopoiesis, myelofibrosis (the people such as James who causes splenomegaly (spleenomegaly) and bone marrow, 2005, Nature 434,1144-1148; People such as Wemig, 2006, Blood.2006 Feb 14, [electronics prior to printing is open]).Clinically, among the CIMF patient existence of mutant with have more aggressive disease relevant with remarkable worse survival rate (people such as Campbell, 2006, Blood, 2098-2100).

This area need be resisted the Phenotype relevant with JAK2 that suddenly change or activatory and is used for the treatment of and relevant myeloproliferative disease and the spinal cord abnormal development syndrome of JAK2 activation.

Summary of the invention

Receptors bind type tyrosine kinase (trk) is a transmembrane protein, and it comprises the outer ligand binding domains of born of the same parents, strides film sequence and cytoplasmic tyrosine kinase domain.Tyrosine kinase plays a role in cell signalling.Cell proliferation, break up, divide a word with a hyphen at the end of a line, example that metabolism and the dead death of program are tyrosine kinase mediated cell response.JAK2 is a nonreceptor tyrosine kinase.

Have been found that the JAK2 inhibitor can be used to treat the constitutive expression of spinal cord hypertrophy disease and other wherein JAK2 to the contributive disease of pathological state.

Therefore the invention provides and use the compositions that comprises the fused pyrrolocarbazoles derivant to treat the method for spinal cord hypertrophy disease and associated conditions.

Spinal cord hypertrophy disease and the associated conditions relevant with activation JAK2 of available method treatment of the present invention include but not limited to myeloproliferative disease, such as, for example polycythemia vera (PV), the thrombocytosis (ET) of the special property sent out, living myelofibrosis (MMM) (also being known as chronic idiopathic myelofibrosis (CIMF)), non-classified spinal cord hypertrophy disease (uMPDs), the eosinophil leucocyte of companion's bone marrow alienation increase syndrome (HES) and systemic mastocytosis (SM).

In the method for the invention, the JAK2 inhibitor of treatment effective dose is used to the experimenter.For average weight is 70 kilograms adult, and dosage regimen can be for example about 20 milligrams about 120 milligrams, twice of every day.In some embodiments, be about 40 milligrams to about 100 milligrams for common adult's dosage, twice of every day.In other embodiments, be about 60 milligrams to about 80 milligrams for common adult's dosage, twice of every day.

In the method for the invention, when having the fused pyrrolocarbazoles derivant, some activity of proteins is compared when not having fused pyrrolocarbazoles and is lowered.These protein include, but are not limited to JAK2, STAT5, STAT3, SHP2, GAB2, AKT and ERK.

Description of drawings

Fig. 1 represents the inhibition of CEP-701 to the JAK2 kinase activity.

Fig. 2 represents the result of allele specific pcr (picture A) and restriction analysis (picture B), described test be in order to confirm in the HEL92 cell, to exist the V617F sudden change.K562 cell (it does not hide this sudden change) is as the wild type contrast.Picture A: for allele specific pcr, the mutant specific primer produces the 203bp product (arrow) of diagnosis sudden change; The 364bp product is as the internal reference of PCR.M, molecular weight marker;

Swimming lane

1 and 2 is to show 364bp mutant and wild-type allele and the special allelic HEL92 cell of 203bp mutant.Swimming lane 3 is the contrast K562 cells that only show the 364bp wild-type allele.Picture B: for restriction analysis, the PCR product that comprises V617F sudden change produces and is digested by BsaXI restriction endonuclease enzyme action from HEL 92 and K562 DNA.Indigested (with "-" expression) and (with "+" expression) PCR product M (labelling of the molecular weight) expression that is digested by BsaXI.Generate the estriction map of the prediction of K562 DNA.Two of each cell line independently DNA prepared products have been tested.Fig. 3 represents the effect that CEP-701 provides JAK2/STAT signal in HEL 92 cells.The CEP-701 of HEL 92 cells and 0.1 μ M, 0.3 μ M, 1.0 μ M and 3.0 μ M cultivated 24 hours, as shown in the figure.To the evaluation of the effect of JAK2/STAT signal granting by as shown in the figure use phosphoric acid specificity STAT3 and the Western blotting of STAT5 antibody carry out the perhaps immunoprecipitation/Western blotting rules IP/WB by JAK2) carry out: total JAK2 antibody is used for IP and estimates phosphorylation by the WB that uses phosphotyrosine antibody.The expression of Bclx1 is measured by Western blotting.

Fig. 4 represents the effect of CEP-701 to the growth of HEL92 cell.As shown in the figure, the cumulative CEP-701 of HEL92 cell and concentration cultivated 24 hours and 48 hours.The effect of cell growth is by the MTS test evaluation.At 10%FCS (picture A and B) or do not have to experimentize in the serum (picture C and D).Picture A system has shown the result who cultivated 24 hours under the condition that 10% hyclone (FBS) exists.Picture B is illustrated under the condition that 10%FBS exists and cultivates 48 hours result.Picture C is illustrated in the result who cultivated under the condition that does not have FBS 24 hours.Screen D is illustrated in the result who cultivated under the condition that does not have FBS 48 hours.For picture A-D, the CEP-701 of cumulative amount is joined (shown in the shade bar diagram) in the independent culture medium; Undressed cell is shown in first bar diagram.

Fig. 5 is illustrated under the condition of per 24 hours additional chemical compounds CEP-701 to the effect of HEL92 cell growth.The HEL92 cell was cultivated 72 hours with the cumulative CEP-701 (as shown in the figure) of concentration in 2%FCS.Per 24 hours additional CEP-701.The effect of cell growth is estimated by the MTS test.Undressed cell is shown in first bar diagram.

Fig. 6 represents the dead inductive effect of CEP-701 pair cell program in the HEL92 cell.The cumulative CEP-701 (as shown in the figure) of HEL92 cell and concentration cultivated 24 hours, 48 hours and 72 hours.Inducing by histone/DNA release test of apoptosis analyzed.Picture A represents test in 24 hours, and picture B represents that test in 48 hours and picture C represent test in 72 hours.

Fig. 7 represent CEP-701 in HEL 92 cells to the activatory inhibition of STAT5.The cumulative CEP-701 of HEL92 cell and concentration cultivates 1 hour, 1.5 hours and 2.5 hours (as shown in the figure).Prepared full cell extract and estimated influence the STAT5 phosphorylation by the Western blotting that uses specific antibody.Bands of a spectrum are carried out quantitatively and with phosphorylation degree carrying out normalization with respect to the total amount of STATS in the sample.The result is expressed as remaining STAT5 phosphorylation and handles the percent that sample is compared with vehicle.

The general value of 5 experiments shows in the bottom.Based on 5 independent experiments, the IC that in the HEL92 cell, STAT5 is suppressed ₅₀Be about 10nM after measured.

Fig. 8 represent CEP-701 in the HEL92 cell to the activatory inhibition of STAT3.The cumulative CEP-701 of HEL92 cell and concentration cultivates 1 hour, 1.5 hours and 2.5 hours (as shown in the figure).Prepared full cell extract and estimated influence the STAT3 phosphorylation by the Western blotting that uses specific antibody.Bands of a spectrum are carried out quantitatively and with phosphorylation degree carrying out normalization with respect to the total amount of STAT3 in the sample.The result is expressed as remaining STAT3 phosphorylation and handles the percent that sample is compared with vehicle.The meansigma methods of 5 experiments shows in the bottom.The IC that in HEL 92 cells, STAT3 is suppressed ₅₀As shown be about 10nM.

Fig. 9 represents the active effect that suppresses of STAT5 that people α 1-AGP mediates CEP-701 in the HEL92 cell.HEL92 cell and 1.0 milligrams/α 1-AGP and the cumulative CEP-701 (as shown in the figure) of concentration cultivated 1 hour, prepared full cell extract and by using the specific antibody Western blotting to estimate influence to the STAT5 phosphorylation.Bands of a spectrum are carried out quantitatively and with phosphorylation degree carrying out normalization with respect to the total amount of STAT5 in the sample.The result is expressed as remaining STAT5 phosphorylation and handles the percent that sample is compared with vehicle.Make the IC that STAT5 is suppressed with the co-cultivation of 1.0 mg/ml AGP ₅₀Change into 3 μ M.

Figure 10 represents the effect of twice subcutaneous CEP-701 of giving every day (30 mg/kg) to the growth of HEL92 tumor xenogeneic graft.Picture A: undressed growth of tumor is represented with square, and treated tumor is represented with triangle.Picture B: the undressed and treated tumor of expression excision.Figure 11 represents the effect that CEP-701 provides the JAK2/STAT signal in HEL 92 tumor xenogeneic grafts.To giving potion CEP-701 (30 mg/kg) under the animal skins of carrying HEL 92 tumor xenogeneic grafts and after CEP-701 gives 0,1,2,4,8 and 12 hour estimating to STAT5 and the activatory effect of STAT3 by Western blotting.Also measured the interior concentration of tumor of CEP-701.Figure 12 represents to be used for to measure by allele specific pcr the genotype of clinical sample of the existence of V617F sudden change.Tested the V617F sudden change of chronic idiopathic myelofibrosis (CIMF) patient #648 and #650 by allele specific pcr.Wild type and mutant allele be as the 364bp fragment amplification, and 203bp (mutant specific PCR product) has shown and has the V617F sudden change.The result who has also shown HEL92 (it comprises mutant allele) and wild type K562 cell.

Figure 13 represents to use the genotype of the clinical sample of restriction endonuclease BstX1.The PCR product that comprises the V617F sudden change carries out enzyme action digestion by BstX1, as shown in the figure.SLC is the irrelevant PCR product of JAK2 that comprises the BstX1 site, the contrast that it is finished as digestion.

Figure 14 represents that CEP-701 is to deriving from the CD34 of patient #648 ⁺The effect of the growth of primary culture (XTT test).Picture A:24 hour.Picture B:48 hour.

Figure 15 represents that CEP-701 is to deriving from the CD34 of patient #648 ⁺The growth of primary culture and the effect of survival.CD34 ⁺Hemopoietic progenitor cell purification from patient #648 obtains and cultivates with the cumulative CEP-701 of concentration, as shown in the figure.Picture A: cell handled 24 hours and estimated by facs analysis the effect of cell growth (living cells) and apoptosis (anchorin (Annexin V)) with CEP-701.Picture B: the cell of handling 24 hours with CEP-701 is diluted in the fresh culture medium and cultivated 72 hours.Cell counting uses trypan blue to measure.Cell number when T=O is illustrated in the experiment beginning.

Figure 16 represents that CEP-701 is at the CD34 that derives from patient #648 ⁺The effect of in the primary culture JAK2/STAT signal being provided.CD34 ⁺Hemopoietic progenitor cell purification from patient #648 obtains and cultivated 1 hour with the cumulative CEP-701 of concentration, as shown in the figure.Expression and the phosphorylation of STAT5, STAT3 that prepared full cell extract and the western blot analysis by using specific antibody.In order to estimate the JAK2 activity,, use the western blot analysis of anti-phosphotyrosine specific antibody (4G10) then with the JAK2 immunoprecipitation.Speckle peeled off and carry out probe once more with JAK2 antibody and survey.Cyclophilin and β actin are as internal reference.

Figure 17 represents that CEP-701 is at the CD34 that derives from patient #648 ⁺In the primary culture to SHP2 and Gab2 activation and effect that AKT and MAPK signal are provided.CD34 ⁺Hemopoietic progenitor cell purification from patient #648 obtains and cultivated 1 hour with the cumulative CEP-701 of concentration, as shown in the figure.The expression and the phosphorylation of multiple signaling molecule that prepared full cell extract and the western blot analysis by using specific antibody.Picture A: estimated CEP-701 to SHP2 and the activatory effect of GAB2 by phosphoric acid specific antibody (pSHP2 and pGAB2).Analyzed total expression level (SHP2 and GAB2).Picture B: use the phosphoric acid specific antibody of antagonism AKT (pAKT) and ERK (pERK) to estimate the effect of CEP-701 to MAPK and the granting of AKT signal.Use specific antibody (AKT and ERK) to analyze total expression level.The β actin is as internal reference.

Figure 18 represents to cultivate 24 hours at the CD34 that derives from patient #648 with CEP-701 ⁺The effect of in the primary culture STAT5 signal being provided.CD34 ⁺Hemopoietic progenitor cell purification from patient #648 obtains and cultivated 24 hours with the cumulative CEP-701 of concentration, as shown in the figure.Prepared full cell extract and the western blot analysis by the using specific antibody expression of STAT5 and the expression of phosphorylation and Bclx1.The expression of β actin and cyclophilin is as internal reference.

The detailed description of exemplary the present invention relates to use condensing of BA Pyrrolo carbazole derivatives treatment multiple illness or the syndromic method relevant with the JAK2 activation. In certain embodiments, illness and/or syndrome comprise for example spinal cord hyperplasia disease (MPDs) and ridge Marrow dysplasia syndrome. Can be used for treating described illness and/or syndromic fused pyrrole and click One of Zole derivatives is indolocarbazole and indeno carbazole derivates. More particularly, the present invention Relate to use indolocarbazole treatment MPDs and the unusual syndrome of spinal cord development and other with The associated conditions that the JAK2 activation is relevant. The fused pyrrolocarbazoles derivative that can be used for this illness Known in the art and can be by many methods preparation well known by persons skilled in the art, institute The method of stating comprises the United States Patent (USP) 4,923,986 such as people such as Murakata; The people's such as Hudkins United States Patent (USP) 5,705,511; The people's such as Hudkins United States Patent (USP) 5,808,060; The people's such as Singh United States Patent (USP) 6,127,401; The people's such as Hudkins United States Patent (USP) 6,841,567; Gingrich etc. People's United States Patent (USP) 6,630,500; The people's such as Lewis United States Patent (USP) 5,756,494; Glicksman United States Patent (USP) 5,468,872 Deng the people; The people's such as Dionne United States Patent (USP) 5,516,771; Hudkins United States Patent (USP) 6,306,849 Deng the people; United States Patent (USP) 6,093,713 with people such as Hudkins; This Openly incorporated in full into this paper as a reference a bit. In certain embodiments, at indolocarbazole Parent material in the preparation for example K-252a and KT-5556 is optically active. Available in preparation In the compound in methods for the treatment of described herein, use initiation material K252a or KT5556 Can cause partially or even wholly transmitting optical activity derives to required fused pyrrolocarbazoles Thing.

As adopted and throughout this disclosure, unless otherwise stated, following term should be managed Solution has following implication.

" pact " used herein refers to the number range of set-point ± 10%. For example, " approximately 20 " comprise 20 ± 10%, or be 18 to 22, comprise end points.

" alkyl " used herein refers to optional substituted, saturated, straight or branched Hydrocarbon (and all combinations and recombinant of the scope in this scope and particular carbon atomicity), the bag Contain about 1 to about 8 carbon atoms, preferably contain about 1 and (be called " low herein to about 4 carbon atoms The level alkyl "). Alkyl includes, but are not limited to methyl, ethyl, n-pro-pyl, isopropyl, positive fourth Base, isobutyl group, the tert-butyl group, n-pentyl, isopentyl, neopentyl, n-hexyl, isohesyl, 3-methylheptyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl.

" thiazolinyl " used herein refers to optional substituted alkyl, has about 2 to about 10 Individual carbon atom, more preferably have about 2 to about 8 carbon atoms and the one or more pairs of keys (and Scope in this scope and all combinations and the recombinant of particular carbon atomicity), wherein alkyl decides Justice as mentioned above.

" alkynyl " used herein refers to optional substituted alkyl, has about 2 to about 10 Individual carbon atom, more preferably have about 2 to about 8 carbon atoms and one or more triple bond (and Scope in this scope and all combinations and the recombinant of particular carbon atomicity), wherein alkyl decides Justice as mentioned above.

" aryl " used herein refers to optional substituted monocyclic, bicyclic or tricyclic fragrance Member ring systems, have about 6 to about 14 carbon atoms (and the scope in this scope and particular carbon are former All combinations and the recombinant of subnumber), more preferably have about 6 to about 10 carbon atoms. Unrestricted The property example comprises for example phenyl, naphthyl, anthryl and phenanthryl.

" aryl alkyl " used herein is meant optional substituted part, it is made up of the alkyl that has aryl substituent, wherein aralkyl moiety has about 7 to about 22 carbon atoms (and all combinations and recombinant of scope in this scope and particular carbon atomic number), preferably has about 7 to about 10 carbon atoms.Non-limitative example comprises for example benzyl, benzhydryl, trityl, phenethyl and two phenethyls." heteroaryl " used herein is meant optional substituted aromatic rings system, has 5 to 14 carboatomic ring members, and wherein one or more carboatomic ring members are replaced by one or more hetero atoms independently.In certain embodiments, hetero atom is selected from S, O or N.Preferably have about altogether 5 to about 14, more preferably from about 5 arrive about 6 carboatomic ring members and heteroatomic ring member's (and all combinations and recombinant of scope in this scope and particular carbon atomic number) heteroaryls.Exemplary heteroaryl includes but not limited to: pyrrole radicals, furyl, pyridine radicals, pyridine-N-oxides, 1,2,4-thiadiazolyl group, pyrimidine radicals, thienyl, isothiazolyl, imidazole radicals, tetrazole radical, pyrazinyl, pyrimidine radicals, quinolyl, isoquinolyl, thienyl, benzothienyl, isobenzofuran-base, pyrazolyl, indyl, purine radicals, carbazyl, benzimidazolyl and different

The azoles base.Heteroaryl can be connected with the remainder of molecule by carbon atom or hetero atom.

" heteroaryl alkyl " used herein is meant optional substituted ring-type system, and it is made up of the alkyl that is replaced by heteroaryl, and wherein heteroaryl and alkyl is described as defined above.Non-limitative example comprises for example 2-(1H-pyrroles-3-yl) ethyl, 3-pyridylmethyl, 5-(2H-tetrazole radical) methyl, and 3-(pyrimidine-2-base)-2-methyl cyclopentane base.

Term used herein " Pro ", " Ser ", " Gly ", " Lys " are meant aminoacid respectively: proline, serine, glycine and lysine.

Term used herein " aminoacid " expression comprises the molecule of amino and carboxyl.Amino acid whose example comprises a-amino acid; That is, by general formula HOOC-CH (NH ₂)-(side chain) Biao Shi carboxylic acid.Amino acid whose side chain comprises natural and non-natural part.The alpha-non-natural amino acid side chain is the part that can be used for replacing the natural amino acid side chain in amino acid analogue for example.For example referring to, Lehninger, Biochemistry, second edition, Worth Publishers, Inc, 1975, the 73-75 pages or leaves, incorporated herein by reference.Preferred a-amino acid comprises glycine, alanine, and proline, glutamic acid and lysine have the D configuration, the L configuration, or be racemic object form.

Just the term of usefulness " GIc " is meant glucose herein.

Term used herein " enrichment in fact ", when being meant stereoisomer or stereogenic centres, be illustrated in the mixture a kind of stereoisomer or a kind of stereogenic centres and exist to preponderate at least about 60%, preferred about 70%, more preferably from about 80%, more preferably from about 90%, more preferably a kind of stereoisomer or a kind of stereoisomers center are at least about 95%.In some preferred embodiments, chemical compound is " mapping is pure in fact ", and that is to say, a kind of stereoisomeric forms in any ratio is to preponderate at least about 97.5%, more preferably from about 99%, more preferably from about 99.5%.

The term that uses in the literary composition " effective dose " is meant a certain amount of of chemical compound as herein described, and it can treat the symptom that is used for suppressing or treating specified disease, disease or the patient's condition effectively.This disease, disease and the patient's condition include, but are not limited to those and the active relevant pathological conditions of JAK2, and wherein treatment comprises for example by suppress the activity of JAK2 with chemical compound exposing cell of the present invention, tissue or receptor.Therefore, for example, term " effective dose " is used for the treatment of the spinal cord hypertrophy when sick when combining with chemical compound of the present invention, be meant treatment usually with relevant symptom, disease, disease and the patient's condition of spinal cord hypertrophy disease.

" pharmaceutically acceptable " used herein is meant in rational medical judgment scope, be fit to the tissue of contact humans and animals and do not have over-drastic toxicity, stimulation, allergy or other problem or complication, and with rational interests/risk than those chemical compounds, material, compositions and/or the dosage form that match.

" officinal salt " used herein is meant the derivant of chemical compound disclosed herein, and wherein parent compound carries out modification by being made into its hydrochlorate or alkali salt.The example of officinal salt includes, but are not limited to the inorganic acid salt or the acylate of alkaline residue such as amine; The alkali salt of acidic residues such as carboxylic acid or organic salt; Or the like.Officinal salt comprises from the nontoxic salts or the quaternary ammonium salt of the routine of the parent compound of for example nontoxic mineral acid or organic acid formation.For example, the nontoxic salts of this routine comprises those salt that derive from mineral acid, and described mineral acid is all example hydrochloric acids, hydrobromic acid, sulphuric acid, sulfamic acid, phosphoric acid and nitric acid; With the salt from the organic acid preparation, described organic acid is such as acetic acid, propanoic acid, succinic acid, hydroxyacetic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-anilinesulfonic acid., 2-acetoxy-benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, ethylenehydrinsulfonic acid; Or the like.The acceptable salt of these physiologys prepares by methods known in the art, for example, in aqueous alcohol free amine base is dissolved in the excessive acid, perhaps uses in alkali metal base such as hydroxide or the amine and the free carboxy acid.Running through chemical compound as herein described can the replacement form be used or prepares.For example, manyly contain form that amino chemical compounds can be used as acid-addition salts and be used or prepare.Usually, these salt have improved the separation property and the operability of chemical compound.For example, according to the difference of reagent, reaction condition etc. and different, chemical compound as herein described can be for example be used or prepares as the form of its hydrochlorate or toluene fulfonate.Isomorphism, all chirality and racemic form, N-oxide, hydrate, solvate and hydrochloride hydrates also covered in the scope of the present invention." hydrate " used herein be meant in molecular forms and the bonded chemical compound of the present invention of water, that is, and wherein not fracture of H-OH key, and can be by formula RH ₂O represents that wherein R is a chemical compound of the present invention.The chemical compound that provides can form and surpass a kind of hydrate, comprises for example monohydrate (RH ₂O) or polyhydrate (RnH ₂O, wherein n is〉1 integer), comprise for example dihydrate (R2H ₂O), trihydrate (R3H ₂O) or the like, or hemihydrate, such as for example R _N/2H ₂O, R _N/3H ₂O, R _N/4H ₂O etc., wherein n is an integer.

" solvate " used herein is meant in molecular forms and the bonded chemical compound of the present invention of solvent; Promptly; Wherein solvent carries out the coordination combination, and can be represented by formula R (solvent); Wherein R is a chemical compound of the present invention.The chemical compound that provides can form and surpass a kind of solvate, comprise for example single solvate (R (solvent)) or multi-solvent compound (Rn (solvent), wherein n is〉1 integer), comprise for example two solvates (R2 (solvent)), three solvates (R3 (solvent)) or the like, or half solvate, such as for example R _N/2(solvent), R _N/3(solvent), R _N/4(solvent) or the like, wherein n is an integer.The solvent of this paper comprises mixed solvent, methanol for example, and therefore, solvate can be incorporated one or more solvents in solvate.

" hydrochloride hydrates " used herein is meant and can combines complex that forms or the complex that combines formation by the chemical compound with one or more acid moieties with at least a chemical compound with one or more alkali parts by the chemical compound with one or more basic moieties with at least a chemical compound with one or more acid moieties, described complex further with water molecules to form hydrate, the described as defined above and R of wherein said hydrate represents the above-described complex of this paper.

Running through chemical compound as herein described can the replacement form be used or prepares.For example, manyly comprise form that amino chemical compounds can be used as acid-addition salts and be used or prepare.Usually, these salt have improved the separation property and the operability of chemical compound.For example, according to the difference of reagent, reaction condition etc. and different, chemical compound as herein described can be for example be used or prepares as the form of its hydrochlorate or toluene fulfonate.Isomorphism, all chirality and racemic form, N-oxide, hydrate, solvate and hydrochloride hydrates also covered in the scope of the present invention.

" patient " used herein is meant animal, and the born of the same parents mammal preferably refers to the people.

" prodrug " used herein is meant the maximized chemical compound of amount that makes the active substance that arrives desired area through being designed for especially, they self common non-activity or have minimum required activity, but be converted into bioactive metabolite by biotransformation.

Term used herein " stereoisomer " is meant to have identical chemical composition, but the different chemical compound of the spatial arrangements of atom or group.

" N-oxide " used herein is meant the oxidized chemical compound that obtains having the quaternary nitrogen of positive formal charge and have the oxygen atom that is connected with nitrogen of negative formal charge of the basic nitrogen atom of heteroaryl ring wherein or tertiary amine.

Term used herein " treatment " comprises prophylactic treatment, the treatment of curing the disease property and/or palliative therapy.

When any variable occurred surpassing one time in any formation or in any form, its definition and its definition in every other appearance in each the appearance had nothing to do.

The combination of substituent group and/or variable can be allowed to, as long as this combination obtains stable chemical compound.

Believe that chemical formula used herein and chemical name correctly and have accurately reflected basic compound.Yet character of the present invention and value not exclusively or partly depend on the theory correction of these formulas.Therefore, be appreciated that, chemical formula used herein and the chemical name that belongs to pairing described chemical compound, be intended to limit the present invention never in any form, comprise it is limited to any specific tautomeric form or is limited to any specific optics or geometric isomer, unless obviously defined this spatial chemistry.

Therefore, in certain embodiments, the present invention relates to treat the method for spinal cord hypertrophy disease, comprise the patient that needs are arranged is given with the JAK2 inhibitor for the treatment of effective dose.In some embodiments, the JAK2 inhibitor is condensed pyrrolocarbazole.In certain embodiments, condensed pyrrolocarbazole is an indolocarbazole, and in other embodiments, it is the indeno carbazole.In specific embodiments, condensed pyrrolocarbazole has the form of structure shown in the following formula I or its stereoisomer or officinal salt:

Formula I

Wherein:

Ring B and F are phenyl or heteroaryl independently;

R ¹Be H independently; Alkyl; Aryl; Aryl alkyl; Heteroaryl; Heteroaryl alkyl;-COR ⁹-OR ¹⁰-CONR ⁷R ⁸-NR ⁷R ⁸-(CH ₂) _pNR ⁷R ⁸-(CH ₂) _pOR ¹⁰-O (CH ₂) _pOR ¹⁰Or-O (CH ₂) _pNR ⁷R ⁸

R ²Be H independently;-SO ₂R ⁹-CO ₂R ⁹-COR ⁹The alkyl that contains 1-8 carbon; The thiazolinyl that contains 2-8 carbon; The alkynyl that contains 2-8 carbon; Or contain the monosaccharide of 5-7 carbon,

Wherein each hydroxyl of monosaccharide is randomly substituted by following group independently: contain 1-4 carbon alkyl, contain the alkyl-carbonyl oxygen base of 2-5 carbon or contain the alkoxyl of 1-4 carbon; With

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

Wherein working as X is CHR ¹⁶Or NR ¹⁶The time, R then ²And R ¹⁶Can be randomly be combined together to form and be connected furan and wherein connect 2 of furan and 5 with 5 randomly respectively by R by its 2 ²⁸And R ²⁹Replace; Be connected 3 of furan by R ¹⁷And R ¹⁸Two replace;

R ³, R ⁴, R ⁵And R ⁶Be H independently; Aryl; Heteroaryl; F; Cl; Br; I;-CN;-CF ₃-NO ₂-OR ¹⁰(CH ₂) _pOR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹-CH ₂OR ¹⁴-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-S (O) yR ¹¹-CO ₂R ⁹-COR ⁹-CONR ⁷R ⁸-CHO;-CH=NOR ¹¹-CH=NR ⁹-CH=NNR ¹²R ¹³-(CH ₂) _pS (O) _yR ⁹-CH ₂SR ¹⁵-CH ₂S (O) _yR ¹⁴-(CH ₂) _pNR ⁷R ⁸-(CH ₂) _pNHR ¹⁴The alkyl that contains 1-8 carbon; The thiazolinyl that contains 2-8 carbon; Or contain the alkynyl of 2-8 carbon;

Wherein alkyl, alkenyl or alkynyl are optional separately independently by 1-3 R ²⁷Replace;

X is:

Contain 1-3 carbon and the optional alkylidene that is replaced by at least one following group: OH ,=O ,=NOR ¹¹, OR ¹¹,-OCOR ⁹,-OCONR ⁷R ⁸,-O (CH ₂) _pNR ⁷R ⁸,-O (CH ₂) _pOR ¹⁰, aryl, aryl alkyl, heteroaryl ,-SO ₂R ⁹-CO ₂R ⁹,-COR ⁹, contain 1-8 carbon alkyl, contain 2-8 carbon thiazolinyl, contain the alkynyl of 2-8 carbon or contain the monosaccharide of 5-7 carbon,

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

-O-;-S (O) _y-; N (R ¹⁶); CHR ¹⁶-CH ₂Z-;-Z-CH ₂-; Or-CH ₂ZCH ₂-;

Wherein Z is C (OR ¹¹) (R ¹¹), O, S, C (=O), C (=NOR ¹¹) or NR ¹¹

Wherein working as X is CHR ¹⁶Or NR ¹⁶The time, R then ²And R ¹⁶Can be randomly be combined together to form and be connected furan and wherein connect 2 of furan and 5 with 5 randomly respectively by R by its 2 ²⁸And R ²⁹Replace; Be connected 3 of furan by R ¹⁷And R ¹⁸Two replace; A ¹And A ²Be independently H ,-OR ¹¹,-SR ¹¹Or-N (R ¹¹) ₂Perhaps be combined together to form=O ,=S or=NR ¹¹Part;

B ¹And B ²Be independently H ,-OR ¹¹,-SR ¹¹Or-N (R ¹¹) ₂Perhaps be combined together to form=O ,=S or=NR ¹¹Part;

Precondition is A ¹And A ², or B ¹And B ²At least one pair of be combined together to form=O;

R ⁷And R ⁸Be H or the alkyl that contains 1-4 carbon independently; Perhaps, form 5-7 unit Heterocyclylalkyl with the nitrogen that it connected;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹¹Be H, the alkyl that contains 1-4 carbon, the aryl that contains 6-10 carbon or heteroaryl independently;

R ¹²And R ¹³Be H, alkyl, the aryl that contains 6-10 carbon or heteroaryl independently; Perhaps, form 5-7 unit Heterocyclylalkyl with the nitrogen that it connected; R ¹⁴Be amino acid whose residue after removing the hydroxyl of decarboxylate independently; R ¹⁵Independently for containing the alkyl of 1-4 carbon;

R ¹⁶Be H, the alkyl that contains 1-4 carbon, the thiazolinyl that contains 2-8 carbon, the alkynyl that contains 2-8 carbon, aryl or heteroaryl independently; Wherein alkyl, thiazolinyl, alkynyl, aryl or heteroaryl are optional separately independently by 1-3 R ²⁷Replace or

When X is CHR ¹⁶Or NR ¹⁶The time, R then ²And R ¹⁶Can be randomly be combined together to form and be connected furan and wherein connect 2 of furan and 5 with 5 randomly respectively by R by its 2 ²⁸And R ²⁹Replace; Be connected 3 of furan by R ¹⁷And R ¹⁸Two replace;

R ¹⁷Independently for OH, contain 1-6 carbon O-just-alkyl or contain the O-acyl group of 2-6 carbon;

R ¹⁸Be H independently; The alkyl that contains 1-4 carbon; CONHC ₆H ₅CH ₂Y,

Wherein Y is OR ¹⁹, SOR ²⁰, NR ²¹R ²²Or SR ²³N ₃CO ₂R ¹⁵S-GIc;

CONR ²⁴R ²⁵CH=NNHCONH ₂CONHOR ¹⁰CH=NOR ¹⁰CH=NNHC (=NH) NH ₂ CH=NN (R ²⁶) ₂Or CH ₂NHCONHR ¹⁶

Perhaps R ¹⁷And R ¹⁸Can randomly be combined together to form-CH ₂NHCO ₂-,-CH ₂OC (CH ₃) ₂O-,=O or-CH ₂N (CH ₃) CO ₂-;

R ¹⁹Independently for H, contain the alkyl of 1-4 carbon or contain the acyl group of 2-5 carbon;

R ²⁰Be the Heterocyclylalkyl that contains alkyl, aryl or the nitrogen atom of 1-4 carbon independently;

R ²¹And R ²²Be H, the alkyl that contains 1-4 carbon, Pro, Ser, Gly, Lys or the acyl group that contains 2-5 carbon independently, precondition is R ²¹And R ²²In have only one for Pro, Ser, Gly, Lys or acyl group;

R ²³Be aryl independently, contain the alkyl of 1-4 carbon or the Heterocyclylalkyl of nitrogen atom;

R ²⁴And R ²⁵Be H independently; The alkyl that contains 1-6 carbon; Phenyl; Or contain the hydroxy alkyl of 1-6 carbon; Perhaps, form 5-7 unit Heterocyclylalkyl with the nitrogen that it connected;

R ²⁶Be aryl independently;

R ²⁷Be aryl independently; Heteroaryl; F; Cl; Br; I;-CN;-NO ₂-OR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹The O-THP trtrahydropyranyl;-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-NHC (=NH) NH ₂-NR ¹⁰SO ₂R ⁹-S (O) _yR ¹¹-CO ₂R ⁹-CONR ⁷R ⁸-CHO;-COR ⁹-CH ₂OR ⁷-CH=NNR ¹²R ¹³-CH=NOR ¹¹-CH=NR ⁹-CH=NNHCH (N=NH) NH ₂-SO ₂NR ¹²R ¹³-P (=O) (OR ¹¹) ₂Or-OR ¹⁴

R ²⁸Independently for contain 1-4 carbon alkyl, contain 1-4 carbon alkoxyl, contain 7-10 carbon aryl alkyl ,-(CH ₂) _pOR ¹⁰,-(CH ₂) _pOC (=O) NR ⁷R ⁸Or-(CH ₂) _pNR ⁷R ⁸

R ²⁹Independently for H, contain 1-4 carbon alkyl, contain 1-4 carbon alkoxyl, contain 7-10 carbon aryl alkyl ,-(CH ₂) _pOR ¹⁰,-(CH ₂) _pOC (=O) NR ⁷R ⁸Or-(CH ₂) _pNR ⁷R ⁸

P is the integer of 1-4; With

Y is 0,1 or 2.

In some embodiments, condensed pyrrolocarbazole has the form of structure shown in the Formula Il or its stereoisomer or officinal salt:

Wherein:

R ¹Be H independently; Alkyl; Phenyl; The aryl alkyl that contains 7-10 carbon; 5-6 unit heteroaryl; Heteroaryl alkyl;-COR ⁹-OR ¹⁰-CONR ⁷R ⁸-NR ⁷R ⁸-(CH ₂) _pNR ⁷R ⁸-(CH ₂) _pOR ¹⁰-O (CH ₂) _pOR ¹⁰Or-O (CH ₂) _pNR ⁷R ⁸

R ³, R ⁴, R ⁵And R ⁶Be H independently; Phenyl; 5-6 unit heteroaryl; F; Cl; Br; I;-CN; CF ₃-NO ₂-OR ¹⁰(CH ₂) _pOR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹-CH ₂OR ¹⁴-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-S (O) _yR ¹¹-CO ₂R ⁹-COR ⁹-CONR ⁷R ⁸-CHO;-CH=NOR ¹¹-CH=NR ⁹-CH=NNR ¹²R ¹³-(CH ₂) _pS (O) _yR ⁹-CH ₂SR ¹⁵-CH ₂S (O) _yR ¹⁴-(CH ₂) _pNR ⁷R ⁸-(CH ₂) _pNHR ¹⁴The alkyl that contains 1-8 carbon; The thiazolinyl that contains 2-8 carbon; Or contain the alkynyl of 2-8 carbon;

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

X is-CH-or N;

A ¹And A ²Be independently H ,-OR ¹¹,-SR ¹¹Or-N (R ¹¹) ₂Perhaps, be combined together to form=O ,=S or=NR ¹¹Part;

B ¹And B ²Be independently H ,-OR ¹¹,-SR ¹¹Or-N (R ¹¹) ₂Perhaps, be combined together to form=O ,=S or=NR ¹¹Part;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹²And R ¹³Be H, alkyl, the aryl that contains 6-10 carbon or heteroaryl independently; Perhaps, form 5-7 unit Heterocyclylalkyl with the nitrogen that it connected;

R ¹⁴Be independently amino acid whose remove decarboxylate after residue;

R ¹⁵Independently for containing the alkyl of 1-4 carbon;

R ¹⁶Be H, the alkyl that contains 1-4 carbon, aryl or heteroaryl independently;

Wherein Y is OR ¹⁹, SOR ²⁰, NR ²¹R ²²Or SR ²³N ₃CO ₂R ¹⁵S-Glc; CONR ²⁴R ²⁵CH=NNHCONH ₂CONHOR ¹⁰CH=NOR ¹⁰CH=NNHC (=NH) NH ₂

CH=NN (R ²⁶) ₂Or CH ₂NHCONHR ¹⁶

Perhaps R ¹⁷And R ¹⁸Randomly be combined together to form-CH ₂NHCO ₂-,-CH ₂OC (CH ₃) ₂O-,=O or-CH ₂N (CH ₃) CO ₂-;

R ²⁶Be aryl independently;

R ²⁷Be aryl independently; Heteroaryl; F; Cl; Br; I;-CN;-NO ₂-OR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹The O-THP trtrahydropyranyl;-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-NHC (=NH) NH ₂-NR ¹⁰SO ₂R ⁹-S (O) _yR ¹¹-CO ₂R ⁹-CONR ⁷R ⁸-CHO;-COR ⁹-CH ₂OR ⁷-CH=NNR ¹²R ¹³-CH=NOR ¹¹-CH=NR ⁹-CH=NNHCH (N=NH) NH ₂-SO ₂NR ¹²R ¹³-PO (OR ¹¹) ₂Or-OR ¹⁴

R ²⁸Independently for contain 1-4 carbon alkyl, contain 1-4 carbon alkoxyl, contain 7-10 carbon aryl alkyl ,-(CH ₂) _pOR ¹⁰-, (CH ₂) _pOC (=O) NR ⁷R ⁸Or-(CH ₂) _pNR ⁷R ⁸

P is the integer of 1-4; With

Y is 0,1 or 2.

In some other embodiment, condensed pyrrolocarbazole has structure shown in the Formula Il I or its stereoisomer or officinal salt:

Wherein:

R ³, R ⁴, R ⁵, R ⁶, R ¹⁷, R ¹⁸, R ²⁸, R ²⁹With the definition of X as described about the definition of formula II chemical compound.In some preferred embodiments, R ³, R ⁴, R ⁵And R ⁶H respectively does for oneself.

In other embodiment of the inventive method, condensed pyrrolocarbazole is the chemical compound with structure shown in the formula III, wherein:

R ³, R ⁴, R ⁵And R ⁶Be H independently; Phenyl; F; Cl;-OR ¹⁰(CH ₂) _pOR ¹⁰-NR ⁷R ⁸-CHO;-(CH ₂) _pNR ⁷R ⁸Or contain the alkyl of 1-8 carbon;

Wherein alkyl is randomly by 1-3 R ²⁷Group replaces;

X is-CH-or N;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁴Be amino acid whose residue after removing the hydroxyl of decarboxylate independently;

R ¹⁸Be H, the alkyl that contains 1-4 carbon, CONHC independently ₆H ₅CH ₂OH; CH ₂OCH ₃CH ₂OC (CH ₃) ₃CH ₂NH ₂CO ₂CH ₃Or CONR ²⁴R ²⁵

P is the integer of 1-4; With

Y is 0,1 or 2.

In some other embodiment, condensed pyrrolocarbazole has structure shown in the formula III, wherein: R ³, R ⁴, R ⁵And R ⁶Be independently of one another H, phenyl, F, Cl ,-OR ¹⁰-NR ⁷R ⁸,-(CH ₂) _pR ⁷R ⁸Or contain the alkyl of 1-8 carbon, wherein alkyl is randomly by 1-3 R ²⁷Group replaces; Or the form of its stereoisomer or officinal salt.

In some preferred embodiment of formula III chemical compound, the indolocarbazole of formula III is that mapping is pure in fact.The indolocarbazole of formula III is that it has some structures in the pure preferred embodiment of mapping in fact therein:

Or its stereoisomer or officinal salt, wherein R ³, R ⁴, R ⁵, R ⁶, R ¹⁷, R ¹⁸, R ²⁸, R ²⁹Described with the definition of X such as the above chemical compound and the definition of the embodiment of formula III about formula II as herein described.In some preferred embodiments, the R of the indolocarbazole of formula III ³, R ⁴, R ⁵And R ⁶H respectively does for oneself.In other embodiments:

R ³, R ⁴, R ⁵And R ⁶Independently for H, Cl, contain 1-4 carbon alkyl ,-OR ¹⁰, CH ₂OR ¹⁰, NR ⁷R ⁸, CH ₂NR ⁷R ⁸Or CONR ⁷R ⁸

R ⁷And R ⁸Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁷Independently for OH or the O-that contains 1-4 carbon just-alkyl;

R ¹⁸Be H, CH independently ₂OH, CO ₂CH ₃, CO ₂CH ₂CH ₃, CO ₂CH ₂CH ₂CH ₃, or CO ₂CH (CH ₃) ₂Perhaps

R ²⁸Be CH independently ₃With

R ²⁹Be H or CH independently ₃

In the other embodiments of formula I chemical compound, condensed pyrrolocarbazole has the form of the structure shown in following formula I-A or its stereoisomer or officinal salt:

Formula I-A

Wherein:

Wherein each hydroxyl of monosaccharide is randomly substituted by following group independently: contain the alkyl of 1-4 carbon, contain the alkyl-carbonyl oxygen base of 2-5 carbon or contain the alkoxyl of 1-4 carbon; With

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

X is CHR ¹⁶Or contain 1-2 carbon and the optional alkylidene that is replaced by at least one following group: OH ,=O ,=NOR ¹¹, OR ¹⁰,-OCOR ⁹,-OCONR ⁷R ⁸,-O (CH ₂) _pNR ⁷R ⁸,-O (CH ₂) _pOR ¹⁰, aryl, aryl alkyl, heteroaryl ,-SO ₂R ⁹-CO ₂R ⁹,-COR ⁹, contain 1-8 carbon alkyl, contain 2-8 carbon thiazolinyl, contain the alkynyl of 2-8 carbon or contain the monosaccharide of 5-7 carbon,

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁵Independently for containing the alkyl of 1-4 carbon;

R ¹⁶Be H, the alkyl that contains 1-4 carbon, aryl or heteroaryl independently; Perhaps working as X is CHR ¹⁶The time, R then ²And R ¹⁶Can be randomly be combined together to form and be connected furan and wherein connect 2 of furan and 5 with 5 randomly respectively by R by its 2 ²⁸And R ²⁹Replace; Be connected 3 of furan by R ¹⁷And R ¹⁸Two replace;

Wherein Y is OR ¹⁹, SOR ²⁰, NR ²¹R ²²Or SR ²³N ₃CO ₂R ¹⁵S-GIc; CONR ²⁴R ²⁵CH=NNHCONH ₂CONHOR ¹⁰CH=NOR ¹⁰CH=NNHC (=NH) NH ₂

CH=NN (R ²⁶) ₂Or CH ₂NHCONHR ¹⁶

R ²⁶Be aryl independently;

R ²⁷Be aryl independently; Heteroaryl; F; Cl; Br; I;-CN;-NO ₂-OR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹The O-THP trtrahydropyranyl;-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-NHC (=NH) NH ₂-NR ¹⁰SO ₂R ⁹-S (O) _yR ¹¹-CO ₂R ⁹-CONR ⁷R ⁸-CHO;-COR ⁹-CH ₂OR ⁷-CH=NNR ¹²R ¹³-CH=NOR ¹¹-CH=NR ⁹-CH-NNHCH (N=NH) NH ₂-SO ₂NR ¹²R ¹³-PO (OR ¹¹) ₂Or-OR ¹⁴

P is the integer of 1-4; With

Y is 0,1 or 2.

In some embodiment of the chemical compound of formula I-A, chemical compound has following structure:

Wherein:

R ²Be independently-CO ₂R ⁹-COR ⁹The alkyl that contains 1-4 carbon; Wherein alkyl is randomly by 1-3 R ²⁷Group replaces;

R ³, R ⁴, R ⁵And R ⁶Be H independently; Phenyl; Cl;-OR ¹⁰(CH ₂) _pOR ¹⁰-NR ⁷R ⁸-(CH ₂) _pNR ⁷R ⁸Or contain the alkyl of 1-6 carbon;

Wherein alkyl is randomly by 1-3 R ²⁷Group replaces;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹¹Independently for H, contain 1-4 carbon alkyl, contain the aryl of 6-10 carbon;

R ¹⁶Independently for H, contain the alkyl of 1-4 carbon;

P is the integer of 1-4; With

Y is 0,1 or 2.

In other embodiment of the chemical compound of formula I-A, chemical compound has following structure:

Wherein:

R ⁷And R ⁸Be H or the alkyl that contains 1-4 carbon independently; Perhaps, form the 5-6 unit Heterocyclylalkyl that randomly comprises other theheterocyclic nitrogen atom together with the nitrogen that it was connected;

R ⁹Independently for containing the alkyl or phenyl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹¹Independently for H, contain the alkyl of 1-4 carbon;

R ²⁷Be phenyl independently; 5-6 unit heteroaryl;-OR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹The O-THP trtrahydropyranyl;-NR ₇R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-NR ¹⁰SO ₂R ⁹-S (O) _yR ¹¹-CO ₂R ⁹-CONR ⁷R ⁸-COR ⁹-CH ₂OR ⁷Or-OR ¹⁴

P is the integer of 1-4; With

Y is 0,1 or 2.

Condensed pyrrolocarbazole in the scope of the invention also can be represented by formula IV.Preferred formula IV derivant is called as compound IV-1 hereinafter to IV-4.The functional deriv formula of representing by formula IV:

Formula IV

Or the form of its stereoisomer or officinal salt, wherein:

(a) R ²And R ¹⁶Be selected from independently of one another: H, contain 1-6 carbon alkyl, contain the hydroxy alkyl of 1-3 carbon and contain the thiazolinyl of 3-6 carbon, precondition is R ²And R ¹⁶Not all be H;

(b) A ¹And A ²All be hydrogen and R ³, R ⁴, R ⁵And R ⁶Be that maximum two of H or they are F independently of one another; Cl; Br; I; NO ₂CN or OH; NHCONHR ⁷, R wherein ⁷Be C ₆H ₅Or containing the alkyl of 1-3 carbon, precondition is R ¹, R ², R ⁵And R ⁶In have only one for NHCONHR ⁷CH ₂OR ⁷The alkyl that contains 1-3 carbon; CH ₂OCONHC ₂H ₅Or NHCO ₂CH ₃With

(c) work as A ¹And A ²When the two combines expression O; R then ³, R ⁴, R ⁵And R ⁶The hydrogen of respectively doing for oneself.

The preferred formula IV chemical compound that uses in several different methods of the present invention is that those compound IV-1 in the table 1 arrive IV-4, and wherein substituent group is as follows.

Table 1

(1) R ³, R ⁴, R ⁵And R ⁶Be hydrogen.A ¹And A ²All be hydrogen.

(2) mixture of the 1.5-1.0 of component I V-2 and IV-3.

Feature of the present invention also is the chemical compound represented by following formula V:

R wherein ⁵Expression halogen, CH ₂OCONHR ¹⁴Or NHCO ₂R ¹⁴(R wherein ¹⁴The expression low alkyl group); R ³The expression hydrogen or halogen; Represent CO with X ₂CH ₃, CH ₂OH or CONHR ¹⁵(R wherein ¹⁵Expression hydrogen, the low alkyl group or the aryl that are replaced by hydroxyl), precondition is to get rid of R ⁵=halogen, R ³=hydrogen and X=CO ₂CH ₃Or CH ₂The combination of OH, and R ⁵=R ³=halogen and X=CO ₂CH ₃Combination, and R ⁵=R ³=Br and X=CONHC ₆H ₅Combination.The stereoisomeric forms in any ratio of formula V chemical compound and officinal salt are applicable in the method for the present invention.In certain embodiments, condensed pyrrolocarbazole has structure shown in the following formula:

Formula VI

Wherein X represents CH ₂S (O) R ¹⁶(R wherein ¹⁶The expression aryl or comprise the heterocyclic radical of nitrogen-atoms), CH ₂SR ¹⁶, CH=NN (R ¹⁷) ₂(R wherein ¹⁷The expression aryl), CH ₂NHCONHR ¹⁸(R wherein ¹⁸Expression low alkyl group or aryl) or CH ₂CO ₂CH ₃The stereoisomeric forms in any ratio of formula VI chemical compound and officinal salt are applicable in the method for the present invention.

Feature of the present invention also is chemical compound or its officinal salt represented by following formula VII:

Formula VII

R wherein ²And R ¹⁶One of be that hydrogen and another are pi-allyl, perhaps they all are pi-allyls.

Chemical compound or its officinal salt that condensed pyrrolocarbazole also has structure shown in the following formula VIII:

Formula VIII

R wherein ⁵Expression CH (SC ₆H ₅) ₂, CH (S-CH ₂CH ₂S-), CH ₂SR ²⁴(R wherein ²⁴Expression benzimidazolyl-2 radicals-Ji, furfuryl group, 2-dimethyl aminoethyl or 1H-1,2,4-triazole-3-yl) or CH=NR ²⁵(R wherein ²⁵Expression pyrrolidine-1-base, pyridine-2-base amino, guanidine radicals, morpholinyl, dimethylamino or 4-methyl piperazine-1-yl).

In other preferred embodiment, condensed pyrrolocarbazole is:

More preferably, above-mentioned condensed pyrrolocarbazole is that mapping is pure in fact.In other embodiments, condensed pyrrolocarbazole is:

Chemical compound with this structure used herein is called " CEP-701 ".

The condensed pyrrolocarbazole that is used for the inventive method can be formulated into pharmaceutical composition by mixing with pharmaceutically useful nontoxic excipient and carrier.This compositions can be produced and be used for parenterai administration, particularly liquid solution or form of suspension; Be used for oral administration, particularly liquid agent, tablet or capsule form; Or intranasal administration, particularly powder, nasal drop or aerosol form.

Said composition can be easily administration and can be in unit dosage forms according to any method preparation known in the art.These methods are for example described in Remington ' s PharmaceuticalSciences (Mack Pub.Co., Easton, Pa., 1980).

The liquid dosage form that is used for oral administration comprises pharmaceutically useful Emulsion, microemulsion, solution, suspension, syrup and elixir.Except reactive compound, liquid dosage form can comprise this area inert diluent commonly used, such as, for example water or other solvent, solubilizing agent and emulsifying agent such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethyl formamide, oils (particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, Semen Maydis oil, germ oil, olive oil, Oleum Ricini and Oleum sesami), glycerol, tetrahydrofurfuryl alcohol, the fatty acid ester of Polyethylene Glycol and sorbitan, and composition thereof.Except inert diluent, Orally administered composition can also comprise auxiliary agent, such as, wetting agent, emulsifying agent and suspending agent, sweeting agent, flavoring agent and fumet.

The solid dosage forms that is used for oral administration comprises capsule, tablet, pill, powder and granule.In this solid dosage forms, reactive compound and at least a inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or following material mix: (a) filler or extender such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binding agent is such as for example carboxy-methyl cellulose alginate, gelatin, polyvinylpyrrolidone, sucrose and arabic gum; (c) wetting agent is such as glycerol, (d) disintegrating agent such as agar, calcium carbonate, potato starch or tapioca, alginic acid, some silicate and sodium carbonate, and (e) the solution blocker is such as paraffin; (f) absorption enhancer such as quaternary ammonium compound; (g) wetting agent is such as for example spermol and glyceryl monostearate; (h) adsorbent such as kaolin and bentonite; (i) lubricant such as Talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate sodium lauryl sulphate, and composition thereof.In capsule, tablet and pill, dosage form also can comprise buffer agent.

The solid composite that also can use similar type uses the excipient such as lactose and high molecular weight polyethylene glycol or the like as the filler in the soft hard gelatin capsule of filling.

The solid composite that also can use similar type uses the excipient such as lactose and high molecular weight polyethylene glycol or the like as the filler in the soft hard gelatin capsule of filling.Reactive compound also can be in the micro-encapsulated form with one or more above-mentioned excipient.In solid dosage forms, reactive compound can mix with at least a inert diluent such as sucrose, lactose or starch.This dosage form also can comprise the other material that is different from inert diluent as common practice, as film-making with lubricator with other film-making auxiliary agent such as magnesium stearate and microcrystalline Cellulose.In capsule, tablet and pill, dosage form also can comprise buffer agent.They can randomly comprise opacifier and suitably independent active component or preferential in certain part of intestinal randomly with the compositions of delayed mode release of active ingredients.The example of spendable embedding composition comprises polymer and wax.

The optimal dose of chemical compound of the present invention can be according to such as the difference of following factor and different: the type of spinal cord hypertrophy disease and progress degree, patient's overall health, patient's age and body weight, the effect and the route of administration of chemical compound.The optimization of chemical compound dosage is in those of ordinary skills' the scope, and provides the value in the scope to comprise any value that drops in this scope at this paper.Be used for the inventive method preparation active component when the concentration in blood plasma be about be effective 1 to about 20uM the time, comprise any value that is in this scope, include but not limited to 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 and 19 μ M.In some preferred embodiments, giving with chemical compound is about 5 to about 15 μ M with the concentration that reaches in blood plasma.In other preferred embodiment, giving with chemical compound is about 7.5 to about 15 μ M with the concentration that reaches in blood plasma.

In certain embodiments of the invention, the dosage of chemical compound be every days 200 microgram/kilogram to about 1 gram/kilogram.More preferably, the dosage of chemical compound be every day 250 micrograms/kg body weight to about 3 mg/kg body weight.More preferably, the dosage of chemical compound be every day about 0.5 mg/kg body weight to about 2.3 mg/kg body weight.More preferably, the dosage of chemical compound be every day about 0.8 mg/kg body weight to about 1.3 mg/kg body weight.

In certain embodiments, for common adult's (about 70 kilograms) daily dose be about 20 milligrams to about 300, or about 40 milligrams to about 250 milligrams, or about 40 milligrams to about 100 milligrams.More preferably, daily dose is about 80 milligrams to about 160 milligrams.Daily dose can give usually with once a day, one day twice, one day three times or one day four times.In some embodiments, dosage regimen is about 20 to about 120 milligrams, one day twice.In other embodiments, dosage regimen is about 20 milligrams to about 100 milligrams, one day twice.In other embodiments, dosage is about 60 milligrams to about 80 milligrams, one day twice.

In the method for the invention, when the JAK2 inhibitor given with therapeutic dose, many other activity of proteins other activity of proteins when not having the JAK2 inhibitor is low under the condition that the JAK2 inhibitor exists.These other protein are for example many following protein: JAK2, STAT5, STAT3, SHP2, GAB2, AKT and ERK.

The present invention is further by following examples explanation.Provide embodiment only to be used for illustration purpose, and be not considered to limit the scope of the invention by any way or content.

Embodiment

Patient's cell and cell line

HEL?92.1.7。The red leukaemia of being of HEL 92.1.7 people is available from TCC (TIB-180), and cell grown in RPMI 1640 culture medium that contain the 2mM L-glutaminate, this culture medium is through overregulating the hyclone that contains 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10mM HEPES and 1.0mM Sodium Pyruvate and 10%, as recommending.CEP-701 or vehicle join in the complete medium according to indication and cultivate.

CD34 ⁺。At Cancer center of the University of Pennsylvania (the University of PennsylvaniaCancer Center), from the patient who is diagnosed as MPDs, collected peripheral blood by the assentment back.By Ficoll gradient centrifugation (Pharmacia) washed corpuscles.Remove mononuclear cell layer and washing.The female pearl of mononuclear cell and anti-CD34 immunity is cultivated and used magnetic cell sorter AutoMacs (Miltenyi Biotech, all use the rules of globules and equipment to carry out according to the recommendation of manufacturer) CD34 ⁺Cell purification.With CD34 ⁺Cell washing and counting.Cell is used when fresh or cell is frozen as the living cells in 10% DMSO.With CD34 ⁺(BIT 9500, and Stem Cell Technologies cultivates in IMDM culture medium Vancouver) comprising 20% serum substitute for cell.Cell and stem cell factor, interleukin-6 and interleukin cultivation were used for initial amplification in 3-5 days.After initial incubation, in culture medium, add erythropoietin.Cell further increased be used for experiment up to obtaining enough cells in 2-5 days.The all cells factor derives from R ﹠amp; D Systems.

Use above-mentioned PCR and restriction digest methods analyst chronic idiopathic myelofibrosis (CIMF) patient's genotype.Find that patient #648 and #650 carry the V617F sudden change and (shown by allele specific pcr, Figure 12) with BsaX1 digestion (Figure 13).In Figure 14, the SCLPCR product is with comparing.When using BsaX1 digestion, 496bp SCL product is cut into 356bp, 110bp and 30bp fragment.

Embodiment 1

CEP-701 is to the inhibition of JAK2 kinase activity

Adopt time-resolved fluorescence (TRF) to detect, in the microtitration plate test, tested the ability of kinase activity that CEP-701 suppresses the JAK2 of baculovirus expression.At first with the high adhesive plate of 96 hole Costar (Corning Costar #3922, Corning, NY) (Tris-buffer saline (TBS) IL) reaches 2 hours for Pierce #31000, Rockford to apply the Neutravidin that contains 10 μ g/mL that is under 37 ℃ with every hole 100 microlitres.Apply the 15-mer peptide substrates that the contains 1 μ g/mL (biotinyl-amino-caproyl-EQEDEPEGDYFEWLE-amide that is under 37 ℃ with every hole 100 microlitres then; Infinity Biotech Research and Resource; Aston PA) reaches other 1 hour.Add JAK2 test mixture (cumulative volume=100 μ L/ holes) then in bread board, it is by 20mM HEPES (pH7.2), 0.2 μ M ATP, 1mM MnCl ₂, 0.1% bovine serum albumin (BSA) and variable concentrations CEP-701 (in DMSO, dilute; Finally be in test 2.5% DMSO) form.The adding enzyme (15ng/ml JAK2 lot number JAK2[318], JA2-2.1) and at room temperature allow reaction to carry out 20 minutes.The detection of phosphorylation product is following to be carried out: PY100 antibody (the PerkinElmer LifeSciences #AD0041 that is added in the Eu-N1 labelling that dilutes with 1:5000 among the TBS that comprises 0.05% soil temperature 20 and 0.25%BSA with every hole 100 microlitres, Boston, MA).At room temperature cultivated 1 hour, add then 100 μ L enhancing solution (PerkinElmer LifeSciences #1244-105, Boston, MA).With the plate gentle agitation and after 30 minutes, use PerkinElmer EnVision 2100 (or 2102) multiple labeling to read the fluorescence that the plate device is measured gained solution.In repeated experiments, produce and suppress curve and be depicted as the curve that suppresses the %-log10 compound concentration.Followingly carry out nonlinear regression among the GraphPad Prism and carry out data analysis by S shape dosage-reply (variable slope) equation is coupled to:

y＝bottom+(top-bottom)/(1+10(log?IC _50-X) ^*Hillslope)

Wherein y is the inhibition under given concentration chemical compound, and x is the logarithm of compound concentration, and bottom is that inhibition % under minimum test compound concentration and top are the inhibition % under the highest test compound concentration.The value of bottom and top is separately fixed at 0 and 100.The IC of single curve will be derived from ₅₀Equalization is also used IC ₅₀The meansigma methods report.Draw the active curve of CEP-701 concentration-JAK2 (Fig. 1).The IC that shows CEP-701 ₅₀Be 0.9 ± 0.2nM.

Embodiment 2

The allele specific pcr of A.Val617Phe sudden change

The genotype of cell line and patient's sample such as E.J. etc. carry out described in (2005) ＂ Acquired mutation ofthe tyrosine kinase JAK2 in human myeloproliferative disorders ＂ Lancet365:1054-106.Use two kinds of methods, comprise allele specific pcr and restriction analysis, determine the state of V617F sudden change.Unless otherwise mentioned, otherwise, use Wizard genomic DNA purification kit (Promega), prepare patient's DNA from granulocyte or mononuclear cell according to the description of manufacturer.Unless otherwise mentioned, otherwise, use the PCR Core test kit that derives from Promega to carry out the PCR reaction according to the description of manufacturer.

In brief, in PCR reaction (58 ℃ of annealing temperatures, 38-44 circulation) in, use common reverse primer (5 '-ctgaatagtcctacagtgttttcagtttca-3 ') (SEQ ID NO:1) and two forward primers that the DNA of 80-200ng is increased.First is a mutant special primer (5 '-agcatttggttttaaattatggagtatatt-3 ') (SEQ ID NO:2), and it comprises intentional mispairing from 3 ' terminal the 3rd nucleotide and produces and show the 203bp product that exists V617F to suddenly change to improve specificity and its.Second forward primer (5 '-atctatagtcatgctgaaagtaggagaaag-3 ') (SEQ ID NO:3) from sudden change with the wild-type allele amplification for the 364bp fragment and as internal reference.The result is as described in Fig. 2 (picture A).Two 203bp products that the HEL92 sample has all shown the amplification of internal reference and derived from mutant allele.K562 is as the wild type contrast.

The test of the genotypic restriction endonuclease base of B.JAK2 causes the metathetical G of V617F → T sudden change to eliminate the restriction site that is used for restriction endonuclease BsaX1 that is present among the wild type JAK2.Therefore, hinder BsaX1 identification V617F sudden change.

In order to estimate the existence of the sudden change in cell line and the patient's sample, the use forward primer (5 '-gggtttcctcagaacgttga-3 ') (SEQ IDN O:4) and reverse primer (5 '-tcattgctttcctttttcacaa-3 ') (SEQ ID NO:5), by PCR (57 ℃, 40-44 circulation) 617 the genomic DNA that comprises JAK2 of 200ng is increased.The 460bp fragment that obtains digested 3-4 hour with BsaX1 and analyzes on 2% agarose gel.Wild-type allele produces the fragment of 241bp, 189bp and 31bp, and mutant allele is intact.Derive from the digestion of dna fragmentation tolerance BsaX1 of the amplification of HEL92 cell, and the amplified fragments that derives from the K562 cell is digested by BsaX1 fully.In order to control finishing of digestion, with the dna fragmentation of people SLC gene in self-contained BsaX site increases and digests with BsaX1.Forward primer (5 '-tcctggggtcttctgtcttg-3 ') (SEQ ID NO:6) and reverse primer (5 '-cctgagaggcaatgggagta-3 ') (SEQ ID NO:7) amplification 496bp SLC product, it is digested the fragment that forms 356bp, 110bp and 30bp.The result is shown in Fig. 2 (picture B).

Embodiment 3

The effect of CEP-701 on cell proliferation

CD34 with purification and amplification ⁺Cell counting and bed board are used for the XTT test in 96 orifice plates.Cell was handled 24-72 hour with JAK2 inhibitor or vehicle, and used XTT reagent (Molecular Probes) to analyze relative cell survival rate according to manufacturer's recommendation.

With the HEL92.1.7 cell (2 * 10 of bed board in 96 orifice plates ⁴Individual/hole) handled 24-72 hour with CEP-701 or vehicle, and measure cell survival by MTS reagent (Promega) according to recommending.

The cell survival rate (Fig. 3, picture A-D) of the HEL 92.1.7 cell that mensuration is handled with CEP-701.In picture A, under the condition that hyclone (FBS) exists and with CEP-701, handle and carry out cultivating in 24 hours the back and recently estimate cell mutually with treated with medicaments gained survival rate not.CEP-701 makes cell survival rate be reduced to about 50% (using the CEP-701 of 3.0 μ M).In picture B, under the condition that hyclone (FBS) exists and with CEP-701, handle and carry out cultivating in 48 hours the back and recently estimate cell mutually with treated with medicaments gained survival rate not.CEP-701 makes cell survival rate be reduced to about 25% (using the CEP-701 of 3.0 μ M).In picture C, under the condition that does not have FBS, carry out cultivation in 24 hours and estimated cell afterwards with CEP-701.CEP-701 makes the cell slip be reduced to about 50% (using the CEP-701 of 3.0 μ M), and it is similar to the figure among the picture A.In screen D, under the condition that does not have FBS, carry out cultivation in 48 hours and estimated cell afterwards with CEP-701.CEP-701 makes the cell slip be reduced to about 25% (using the CEP-701 of 3.0 μ M), and it is similar to the figure among the picture B.

Fig. 5 represents the effect of CEP-701 to the survival rate of the hel cell of growing under the condition that has hyclone (FCS) and per 24 hours complementary medicines.In this experiment, HEL 92.1.7 cellular exposure was assigned 72 hours and estimate survival rate in MTS test at medicine.CEP-701 is reduced to cell survival rate to be lower than 5% (using the CEP-701 of 1.0 μ M).

MTS test shows that CEP-701 suppresses to carry the growth of the HEL92 cell of V617F sudden change.

Fig. 6 is illustrated in HEL 92 cells, and the CEP-701 that measures by histone/DNA release test is to the effect of inducing cell program death.This test shows the death of CEP-701 inducing cell program.

Embodiment 4

CEP-701 is to the analysis of the effect of signal path

The full cell extract that uses specific antibody and prepare from patient's sample of cell line and cultivation has been estimated the effect of CEP-701 to signal path.The full cell extract of preparation in following cell lysis buffer solution: (20mM Tris-HCl (pH7.5), 150mM NaCl, 1mMNa ₂EDTA, 1mM EGTA, 1% Triton, 2.5mM tetrasodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na ₃VO ₄, 1 μ g/ml leupeptin), be supplemented with protease inhibitor (CompleteMini, Roche Diagnostic).Measured protein concentration and with equivalent (10-30 μ g/ swimming lane) on the SDS-PAGE gel separately.Protein transduction is moved on in the nitrocellulose filter and uses according to the recommendation of manufacturer the level of specific antibody evaluation expression and/or phosphorylation.Use Supersignal West Pico System (Pierce) that speckle is developed the color according to the description of manufacturer.Measure the activation of JAK2 by immunoprecipitation/Western blotting rules.

In brief, according to recommend using immunoprecipitation Starter Pack (AmershamBiosciences), from the extract of the JAK2 antibody of 250 μ g, make JAK2 that immunoprecipitation take place.Behind Western blotting, use phosphotyrosine specific antibody 4G10 (Upstate) to carry out probe and survey the tyrosine phosphorylation of estimating JAK2.

The cumulative CEP-701 (CEP-701 of from 0.1 to 3.0 μ M) of Fig. 3 picture A indicated concentration is to the dose-dependent inhibition of STAT5 phosphorylation.The cumulative CEP-701 (from 0.03 to 1.0 μ M) of picture B indicated concentration is to the inhibition of the phosphorylation of JAK2 and STAT3.Picture B also represents BCIX _LThe dose-dependent inhibition of the expression of (the downstream effect device that the JAK2/STAT5 signal is provided).

Fig. 7 is illustrated in and is exposed under the cumulative CEP-701 of concentration after 1.0,1.5 and 2.5 hours the inhibition of the phosphorylation of STAT5.The bottom that on average is suppressed at of 5 experiments shows.The IC that in the HEL92.1.7 cell, STAT5 is suppressed ₅₀Be about 10nM, it will be interpreted as having the inhibition to the granting of JAK2/STAT signal on the clinical meaning.Fig. 8 is illustrated in and is exposed under the cumulative CEP-701 of concentration after 1.0,1.5 and 2.5 hours the inhibition of the phosphorylation of STAT3.The bottom that on average is suppressed at of 5 experiments shows.The IC that in HEL 92.1.7 cell, suppresses for STAT3 ₅₀Be about 10nM, it will be interpreted as having the inhibition that the JAK2/STAT signal on the clinical meaning is provided.

Fig. 9 is illustrated in the HEL92 cell, and α 1-AGP is to the effect of the active inhibition of STAT5 of CEP-701 mediation.As described in Figure 9, the α 1-AGP of 1.0 mg/ml makes the IC that STAT5 is suppressed ₅₀Change into 3 μ M.These concentration for example can easily realize in 80 milligrams of dosage regimens in vivo.

Embodiment 5

CEP-701 is to the effect of the growth of HEL 92 tumor xenogeneic grafts.

In the mice of carrying HEL 92 xenografts, estimate the effect of CEP-701 (comparing) to the tumor growth with the vehicle contrast.In brief, twice of every day, the subcutaneous CEP-701 that gives with 30 mg/kg and estimated gross tumor volume under selected natural law shown in Figure 10.The tumor growth is suppressed by CEP-701, and the gross tumor volume in the mice of handling with CEP-701 significantly reduces.

Also in the HEL92 xenograft, estimated the effect that CEP-701 provides the JAK2/STAT signal.In this research, carry the HEL.92 tumor animals received potion CEP-701 (30 mg/kg s.c.), and have been estimated the kinetics that STAT5 and STAT3 suppress by Western blotting at different time points.As shown in figure 11, CEP-701 suppresses STAT5 and STAT3 phosphorylation consumingly, observes maximum the inhibition after administration in 2-4 hour.It is relevant well with the interior CEP-701 concentration of tumor to suppress kinetics, has confirmed that further CEP-701 suppresses the ability that the JAK2/STAT signal is provided in vivo.

Embodiment 6

Patient's sample

Shown that in XTT test (Figure 14) CEP-701 suppresses from the isolated CD34 of patient #648 ⁺The growth of cell.The cumulative CEP-701 (0.03 μ M, 0.1 μ M and 0.3 μ M) of concentration reduces the cell growth at 24 hours (picture A) and 48 hours (picture B).

Figure 15 represents the CD34 that derives from patient #648 with the CEP-701 processing ⁺The fluorescence activated cell sorter of primary culture (FACS) is analyzed.In picture A, white bar diagram shows the number of living cells.Inducing of black bar diagram showed cell program death among the picture A shows by measuring anchorin.In the picture B of Figure 15, the black bar diagram shows the CD34 that lives ⁺The number of cell, it also was diluted in the fresh culture medium then in 24 hours with the cumulative CEP-701 processing of concentration and cultivated other 72 hours.Use trypan blue to measure cell counting.

Figure 16 represents that CEP-701 is at the CD34 that derives from patient #648 ⁺The effect of in the primary culture JAK2/STAT signal being provided.CD34 ⁺Hemopoietic progenitor cell purification and cultivated as shown in the figure 1 hour from patient #648 with the CEP-701 of concentration cumulative (30nM, 100nM, 300nM and 1000nM).Use expression and the phosphorylation of specific antibody by western blot analysis, STAT3.In order to estimate the JAK2 activity,, use anti-phosphotyrosine specific antibody (4G10) to carry out western blot analysis then with the JAK2 immunoprecipitation.The CD34 that V617F suddenlys change and derives from MPD patient that carries that is cultivating ⁺In the precursor, CEP-701 shows the granting of inhibition JAK2/STAT signal.

The CD34 that derives from patient #648 that is cultivating ⁺Estimated the activatory effect of CEP-701 in the cell to SHP2, GAB2, AKT and ERK.Use Western blotting to detect phosphorylation and the expression of SHP2, GAB2 (Figure 17 picture A) and AKT and ERK (Figure 17 picture B).The CEP-701 that concentration is cumulative joins the CD34 that derives from patient #648 ⁺In the primary culture of cell.The concentration of using is 0 (that is, having only vehicle), 30,100,300 and the CEP-701 of 1000nM.CEP-701 shows that inhibition involves CD34 ⁺Many signals of the propagation of precursor and survival are provided the path, have hinted the extensive inhibition under acceptor levels in the cell that carries the V617F sudden change.In picture B, the expression of house-keeping gene beta-actin is with comparing.

Also at expression and phosphorylation and the BCL-X of detection as the STAT5 of the downstream targets of JAK2/STAT signal granting _LThe test of expression in estimated the cell (Figure 18) that derives from patient #648.The CEP-701 that concentration is cumulative joins the CD34 that derives from patient #648 ⁺In the primary culture of cell.The concentration of using is 0 (that is, having only vehicle), 30,100,300 and the CEP-701 of 1000nM.The expression of house-keeping gene beta-actin and cyclophilin is with comparing.In these cells, CEP-701 shows that inhibition JAK2/STAT signal is provided and Bclx1 expresses.Prolong and be exposed to the downward modulation that (for example multidose administration) can cause STAT5 to express under the CEP-701.

One skilled in the art can appreciate that and to carry out multiple improvement and change according to above-mentioned instruction to the present invention.Therefore, will be understood that in the scope that is defined by the claims, the present invention can be put into practice with the different modes that this paper is specified, and scope of the present invention is intended to contain these all variants.

Claims

1. treat the method for spinal cord hypertrophy disease, this method comprises gives with the chemical compound as the JAK2 inhibitor for the treatment of effective dose the patient.

2. the process of claim 1 wherein that the JAK2 inhibitor is condensed pyrrolocarbazole.

3. treat the method for spinal cord hypertrophy disease, this method comprises gives with the chemical compound shown in the following formula of treatment effective dose or the form of its stereoisomer or officinal salt the patient,

Wherein:

Ring B and F are phenyl or heteroaryl independently;

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

R ³, R ⁴, R ⁵And R ⁶Be H independently; Aryl; Heteroaryl; F; Cl; Br; I;-CN;-CF ₃-NO ₂-OR ¹⁰(CH ₂) _pOR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹-CH ₂OR ¹⁴-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-S (O) _yR ¹¹-CO ₂R ⁹-COR ⁹-CONR ⁷R ⁸-CHO;-CH=NOR ¹¹-CH=NR ⁹-CH=NNR ¹²R ¹³-(CH ₂) _pS (O) _yR ⁹-CH ₂SR ¹⁵-CH ₂S (O) _yR ¹⁴-(CH ₂) _pNR ⁷R ⁸-(CH ₂) _pNHR ¹⁴The alkyl that contains 1-8 carbon; The thiazolinyl that contains 2-8 carbon; Or contain the alkynyl of 2-8 carbon;

X is:

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

-O-;-S (O) _y-; N (R ¹⁶); CHR ¹⁶-CH ₂Z-;-Z-CH ₂-; Or-CH ₂ZCH ₂-;

Wherein Z is C (OR ¹¹) (R ¹¹), O, S, C (=O), C (=NOR ¹¹) or NR ¹¹

A ¹And A ²Be independently H ,-OR ¹¹,-SR ¹¹Or-N (R ¹¹) ₂Perhaps be combined together to form=O ,=S or=NR ¹¹Part;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁵Independently for containing the alkyl of 1-4 carbon;

CH=NN (R ²⁶) ₂Or CH ₂NHCONHR ¹⁶

R ²⁶Be aryl independently;

P is the integer of 1-4; With

Y is 0,1 or 2.

4. the method for claim 3, wherein chemical compound has the form of the structure shown in the following formula or its stereoisomer or officinal salt:

Wherein:

Wherein alkyl, alkenyl or alkynyl are randomly by 1-3 R ²⁷Group replaces;

X is-CH-or N;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁴Be independently amino acid whose remove decarboxylate after residue;

R ¹⁵Independently for containing the alkyl of 1-4 carbon;

CH=NN (R ²⁶) ₂Or CH ₂NHCONHR ¹⁶

R ²⁶Be aryl independently;

P is the integer of 1-4; With

Y is 0,1 or 2.

5. the method for claim 4, wherein chemical compound has the form of structure shown in the following formula or its stereoisomer or officinal salt:

Wherein:

Wherein alkyl is randomly by 1-3 R ²⁷Group replaces;

X is-CH-or N;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

P is the integer of 1-4; With

Y is 0,1 or 2.

6. the method for claim 5, wherein R ³, R ⁴, R ⁵And R ⁶Be independently of one another H, phenyl, F, Cl ,-OR ¹⁰,-NR ⁷R ⁸,-CHO ,-(CH ₂) _pR ⁷R ⁸Or contain the alkyl of 1-8 carbon, wherein alkyl is randomly by 1-3 R ²⁷Group replaces.

7. the method for claim 4, wherein chemical compound has the form of structure shown in the following formula or its stereoisomer or officinal salt:

Wherein:

R ⁷And R ⁸Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁷Independently for OH or the O-that contains 1-4 carbon just-alkyl;

R ²⁸Be CH independently ₃With

R ²⁹Be H or CH independently ₃

8. the method for claim 7, wherein chemical compound has structure shown in the following formula:

9. the method for claim 7, wherein chemical compound has the form of structure shown in the following formula or its stereoisomer or officinal salt:

Wherein:

(b) A ¹And A ²All be hydrogen and R ³, R ⁴, R ⁵And R ⁶Be that maximum two of H or they are F independently of one another; Cl; Br; I; NO ₂CN or OH; NHCONHR ⁷, R wherein ⁷For containing the alkyl of 1-4 carbon, precondition is R ³, R ⁴, R ⁵And R ⁶In have only one for NHCONHR ⁷CH ₂OR ¹⁰The alkyl that contains 1-4 carbon; CH ₂OCONHC ₂H ₅Or NHCO ₂CH ₃With

10. the method for claim 3, wherein chemical compound has the form of following formula structure or its stereoisomer or officinal salt:

Wherein:

Wherein alkyl is randomly by 1-3 R ²⁷Group replaces;

R ⁹Independently for containing alkyl, aryl or the heteroaryl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹⁶Independently for H, contain the alkyl of 1-4 carbon;

P is the integer of 1-4; With

Y is 0,1 or 2.

11. the method for claim 10, wherein chemical compound has the form of following formula structure or its stereoisomer or officinal salt:

Wherein:

R ⁹Independently for containing the alkyl or phenyl of 1-4 carbon;

R ¹⁰Be H or the alkyl that contains 1-4 carbon independently;

R ¹¹Independently for H, contain the alkyl of 1-4 carbon;

R ²⁷Be phenyl independently; 5-6 unit heteroaryl;-OR ¹⁰-O (CH ₂) _pNR ⁷R ⁸-OCOR ⁹-OCONHR ⁹The O-THP trtrahydropyranyl;-NR ⁷R ⁸-NR ¹⁰COR ⁹-NR ¹⁰CO ₂R ⁹-NR ¹⁰CONR ⁷R ⁸-NR ¹⁰SO ₂R ⁹-S (O) _yR ¹¹-CO ₂R ⁹-CONR ⁷R ⁸-COR ⁹-CH ₂OR ⁷Or-OR ¹⁴

P is the integer of 1-4; With

Y is 0,1 or 2.

12. the method for claim 3, wherein said spinal cord hypertrophy disease is selected from: myelofibrosis (MMM), idiopathic myelofibrosis (CIMF), non-classified spinal cord hypertrophy disease (uMPDs), eosinophil leucocyte that polycythemia vera (PV), the thrombocytosis (ET) of the special property sent out, companion's bone marrow alienation are given birth to increase syndrome (HES) and systemic mastocytosis (SM).

13. the method for claim 8, wherein said spinal cord hypertrophy disease is selected from: myelofibrosis (MMM), idiopathic myelofibrosis (CIMF), non-classified spinal cord hypertrophy disease (uMPDs), eosinophil leucocyte that polycythemia vera (PV), the thrombocytosis (ET) of the special property sent out, companion's bone marrow alienation are given birth to increase syndrome (HES) and systemic mastocytosis (SM).

14. the method for claim 13, wherein chemical compound with every day about 0.8 mg/kg body weight give to the amount of about 1.3 mg/kg body weight and use.

15. the method for claim 13, wherein chemical compound is given for twice to about 80 milligrams amount every day with about 60 milligrams and is used.