WO2020135489A1 - 2,4-二氨基嘧啶衍生物 - Google Patents

2,4-二氨基嘧啶衍生物 Download PDF

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WO2020135489A1
WO2020135489A1 PCT/CN2019/128246 CN2019128246W WO2020135489A1 WO 2020135489 A1 WO2020135489 A1 WO 2020135489A1 CN 2019128246 W CN2019128246 W CN 2019128246W WO 2020135489 A1 WO2020135489 A1 WO 2020135489A1
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alkyl
halogen
heteroalkyl
alkynyl
alkenyl
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PCT/CN2019/128246
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English (en)
French (fr)
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张磊
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上海喆邺生物科技有限公司
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Priority to CN201980086260.6A priority Critical patent/CN113227080B/zh
Publication of WO2020135489A1 publication Critical patent/WO2020135489A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the present invention relates to a class of 2,4-diaminopyrimidine derivatives, their pharmaceutically acceptable salts, hydrates, solvates or stereoisomers, and their preparation methods and the use of such compounds alone or in combination with other drugs Use in the treatment of diseases with ALK2 kinase-mediated pathological features.
  • Hepcidin reduces duodenal iron absorption and iron export from monocytes/macrophages by binding and inducing internalization and degradation of membrane ferroportin (FPN1) (Hentze MW. et al., Cell. 2010; 142 (1): 24-38; Nemeth E. et al., Science. 2004; 306(5704): 2090–2093; Theurl I. et al., Blood. 2009; 113(21): 5277-5286). Therefore, elevated serum hepcidin levels enhance the storage of iron in the reticuloendothelial system and lead to a decrease in available iron and the production of iron-deficiency erythrocytes.
  • FPN1 membrane ferroportin
  • Hepcidin can cause severe functional iron-deficiency anemia in humans, and is the core of the pathophysiology of anemia of chronic disease (ACD) (Weiss G. et al, N Engl J Med. 2005; 352(10): 1011-1023 ). Hepcidin's transcription in the liver is mainly affected by several factors, including the body's iron storage, the iron demand for erythropoiesis, hypoxia and inflammation. Bone morphogenetic proteins (BMPs) play an important role in mediating these factors and driving transcriptional induction of hepcidin by activating BMP receptor (BMPR)-SMAD signaling (Wang RH. et al., Cell Metab.
  • BMPs BMP receptor
  • BMPR activin receptor-like kinase 2 ALK2
  • ALK3 BMPR activin receptor-like kinase 2
  • liver-specific deletion of ALK2 or ALK3 blocks the induction of BMP ligand binding downstream hepcidin production and leads to iron in mice Overload (Steinbicker AU. et al., Blood. 2011; 118(15): 4224–4230).
  • BMP ligands interact with bone morphogenetic protein receptors (BMPRs), which belong to the serine/threonine kinase receptor of the TGF- ⁇ superfamily.
  • Anemia caused by chronic infection, inflammation and malignant tumors is usually called anemia of chronic disease.
  • BMP signaling induces hepcidin liver expression through two BMP type I receptors, ALK2 and ALK3. Hepcidin degrades iron output, iron transporters reduce blood iron levels, leading to increased iron storage in macrophages and other cells, and making it unusable for hemoglobin and red blood cell function.
  • ACD anemia of chronic disease
  • Blocking BMP signaling is a potential therapy for the treatment of anemia in chronic diseases.
  • the BMP signaling pathway also plays an important role in the growth and deterioration of tumor cells, especially breast cancer, prostate cancer, and other tumors that are prone to bone metastasis (Lin Ye. et al., Front Biosci. 2011(16):865-897) .
  • the expression of BMPs and BMPRs in metastatic breast cancer is higher than that without metastasis, and it is also highly expressed in prostate cancer with bone metastasis.
  • the object of the present invention is to provide a compound represented by formula (I) or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof,
  • X is -(CR 7 R 8 ) p -;
  • R 1 is selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -(CH 2 ) q NR 9 R 10 -, -CONR 9 R 10 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, heteroalkyl, 3-8 membered cycloalkyl, saturated or unsaturated heterocyclic group, aromatic Group or heteroaryl, wherein alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl can be optionally substituted;
  • R 2 is selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -(CH 2 ) q NR 9 R 10 -, -CONR 9 R 10 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, heteroalkyl, -OR 9 , -COR 9 , -COOR 9 or -NR 9 COR 10 , Wherein alkyl, alkenyl, alkynyl or heteroalkyl can be optionally substituted;
  • R 3 , R 4 and R 5 are independently selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -(CH 2 ) q NR 9 R 10 -, -CONR 9 R 10 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, heteroalkyl, -OR 9 , -COR 9 ,- COOR 9 , -SO 2 R 9 , -NR 9 COR 10 , -NR 9 CONR 10 R 11 , -NR 9 CO 2 R 10 , -NR 9 SO 2 NR 10 R 11 or -NR 9 SO 2 R 10 , where Alkyl, alkenyl, alkynyl or heteroalkyl can be optionally substituted;
  • R 6 is selected from -H, halogen, -OH, -CN, -CF 3 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl or heteroalkyl , Where alkyl, alkenyl, alkynyl or heteroalkyl can be optionally substituted;
  • R 7 and R 8 are independently selected from -H, halogen, -C 1-6 alkyl, heteroalkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, saturated or unsaturated heterocycle Group, oxo or 3-8 membered cycloalkyl; wherein alkyl, alkenyl, alkynyl, heterocyclic or cycloalkyl can be optionally substituted;
  • R 9 , R 10 and R 11 are each independently selected from -H, heteroalkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, 3-8 membered cycloalkane Group, saturated or unsaturated heterocyclic group, aryl group or heteroaryl group; wherein (R 9 and R 10 ) and/or (R 10 and R 11 ) together with the nitrogen atom to which they are attached may form at least one R 12 substituted saturated or unsaturated heterocycle;
  • Each R 12 is independently selected from -H, halogen, -C 1-6 alkyl or oxo;
  • n 0, 1, 2 or 3;
  • p 1, 2 or 3;
  • q 0, 1, 2 or 3.
  • the invention provides compounds of formula (II),
  • X is -(CR 7 R 8 ) p -;
  • R 1 is selected from aryl or heteroaryl which may be optionally substituted
  • R 2 is selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -(CH 2 ) q NR 9 R 10 -, -CONR 9 R 10 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, heteroalkyl, -OR 9 , -COR 9 , -COOR 9 or -NR 9 COR 10 , Wherein alkyl, alkenyl, alkynyl or heteroalkyl can be optionally substituted;
  • R 3 , R 4 and R 5 are independently selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -(CH 2 ) q NR 9 R 10 -, -CONR 9 R 10 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, heteroalkyl, -OR 9 , -COR 9 ,- COOR 9 , -SO 2 R 9 , -NR 9 COR 10 , -NR 9 CONR 10 R 11 , -NR 9 CO 2 R 10 , -NR 9 SO 2 NR 10 R 11 or -NR 9 SO 2 R 10 , where Alkyl, alkenyl, alkynyl or heteroalkyl can be optionally substituted;
  • R 6 is selected from -H, halogen, -OH, -CN, -CF 3 , -NO 2 , -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl or heteroalkyl , Where alkyl, alkenyl, alkynyl or heteroalkyl can be optionally substituted;
  • R 7 and R 8 are independently selected from -H, halogen, -C 1-6 alkyl, heteroalkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, saturated or unsaturated heterocycle Group, oxo or 3-8 membered cycloalkyl; wherein alkyl, alkenyl, alkynyl, heterocyclic or cycloalkyl can be optionally substituted;
  • R 9 , R 10 and R 11 are each independently selected from -H, heteroalkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, 3-8 membered cycloalkane Group, saturated or unsaturated heterocyclic group, aryl group or heteroaryl group; wherein (R 9 and R 10 ) and/or (R 10 and R 11 ) together with the nitrogen atom to which they are attached may form at least one R 12 substituted saturated or unsaturated heterocycle;
  • Each R 12 is independently selected from -H, halogen, -C 1-6 alkyl or oxo;
  • p 1, 2 or 3;
  • q 0, 1, 2 or 3.
  • the invention provides compounds of formula (III),
  • X is -(CR 7 R 8 ) p -;
  • R 1 is selected from optionally substituted heteroaryl
  • R 2 is selected from -H, halogen or -C 1-6 alkyl; wherein the alkyl group may be substituted with a substituent selected from -H, halogen, -C 1-4 alkyl or oxo;
  • R 3 and R 4 are independently selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -(CH 2 ) q NR 9 R 10 -, -CONR 9 R 10 , -NO 2 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, heteroalkyl, -OR 9 , -COR 9 , -COOR 9 , -SO 2 R 9 , -NR 9 COR 10 , -NR 9 CONR 10 R 11 , -NR 9 CO 2 R 10 , -NR 9 SO 2 NR 10 R 11 or -NR 9 SO 2 R 10 , wherein alkyl, alkenyl, Alkynyl or heteroalkyl can be optionally substituted;
  • R 6 is selected from —H, halogen, —OH, —CN, —CF 3 , —NO 2 , —C 1-6 alkyl or heteroalkyl, wherein the alkyl or heteroalkyl can be optionally substituted;
  • R 7 and R 8 are independently selected from -H, halogen, -C 1-6 alkyl, heteroalkyl, -C 2-6 alkenyl, -C 2-6 alkynyl or oxo; wherein alkyl, The alkenyl or alkynyl group may be substituted with a substituent selected from -H, halogen, -C 1-4 alkyl;
  • R 9 , R 10 and R 11 are each independently selected from -H, heteroalkyl, -C 1-6 alkyl, -C 2-6 alkenyl, -C 2-6 alkynyl, 3-8 membered cycloalkane Group, saturated or unsaturated heterocyclic group, aryl group or heteroaryl group; wherein (R 9 and R 10 ) and/or (R 10 and R 11 ) together with the nitrogen atom to which they are attached may form at least one R 12 substituted saturated or unsaturated heterocycle;
  • Each R 12 is independently selected from -H, halogen, -C 1-6 alkyl or oxo;
  • p 1, 2 or 3;
  • q 0, 1, 2 or 3.
  • the present invention provides a compound of formula (III), a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, wherein:
  • X is -(CR 7 R 8 ) p -;
  • R 1 is selected from pyridyl which may be optionally substituted
  • R 2 is selected from -H, halogen or -C 1-6 alkyl; wherein the alkyl group may be substituted with a substituent selected from -H, halogen, -C 1-4 alkyl or oxo;
  • R 3 and R 4 are independently selected from -H, halogen, -C 1-6 alkyl or heteroalkyl; wherein alkyl or heteroalkyl can be selected from -H, halogen, -C 1-4 alkyl Or oxo substituent substitution;
  • R 6 is selected from -H, halogen, -OH, -CN, -C 1-6 alkyl or -C 1-6 alkoxy; wherein alkyl or alkoxy can be selected from -H, halogen, -C 1-4 alkyl or oxo substituent substitution;
  • R 7 and R 8 are independently selected from -H, halogen, -C 1-6 alkyl or -C 1-6 alkoxy, wherein the alkyl or alkoxy group may be selected from -H, halogen or -C 1-4 alkyl substituent substitution;
  • p 1 or 2.
  • the present invention provides a compound of formula (III), a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, wherein:
  • X is -(CR 7 R 8 ) p -;
  • R 1 is Wherein R 13 is selected from -H, halogen, -OH, -CN, -CF 3 , -CHF 2 , -CH 2 F, -OCF 3 , -NO 2 , -NH 2 , -C 1-6 alkyl,- C 2-6 alkenyl, -C 2-6 alkynyl or heteroalkyl;
  • R 2 is selected from -H, halogen or C 1-6 alkyl
  • R 3 and R 4 are independently selected from -H, halogen, C 1-6 alkyl or -C 1-6 alkoxy, wherein the alkyl or alkoxy group may be selected from -H, halogen or -C 1 -4 alkyl or oxo substituent substitution;
  • R 6 is selected from -H, halogen, -OH, -CN, -C 1-6 alkyl or -C 1-6 alkoxy;
  • R 7 and R 8 are each independently selected from -H, halogen or -C 1-6 alkyl, wherein the alkyl group may be substituted with a substituent selected from -H, halogen or -C 1-4 alkyl;
  • p 1 or 2.
  • the present invention provides a compound of formula (III), a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, wherein:
  • X is -(CR 7 R 8 ) p -;
  • R 1 is Wherein R 13 is selected from -H, halogen or -C 1-4 alkyl;
  • R 2 is selected from -H or -C 1-4 alkyl
  • R 3 is selected from -H, -C 1-4 alkyl or -C 1-4 alkoxy
  • R 4 is selected from -H or -C 1-4 alkyl
  • R 6 is selected from -H, halogen, -CN, -C 1-4 alkyl or -C 1-4 alkoxy;
  • R 7 and R 8 are each independently selected from -H or -C 1-4 alkyl
  • p 1 or 2.
  • the present invention relates to typical compounds of formula (I)-formula (III) as follows, but not limited to the following compounds:
  • halogen refers herein to -F, -Cl, -Br and -I.
  • alkyl refers herein to an alkyl group that does not contain heteroatoms. Therefore, the term includes linear alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.
  • the term also includes branched chain isomers of branched alkyl groups, including but not limited to groups such as -CH(CH 3 ) 2 , -CH(CH 3 )(CH 2 CH 3 ), -CH(CH 2 CH 3 ) 2 , -C(CH 3 ) 3 , -C(CH 2 CH 3 ) 3 , -CH 2 CH(CH 3 ) 2 , -CH 2 CH(CH 3 )(CH 2 CH 3 ),- CH 2 CH(CH 2 CH 3 ) 2 , -CH 2 C(CH 3 ) 3 , -CH 2 C(CH 2 CH 3 ) 3 , -CH(CH 3 )-CH(CH 3 )(CH 2 CH 3 ), -CH 2 CH 2 CH(CH 3 ) 2 , -CH 2 CH 2 CH(CH 3 )(CH 2 CH 3 ), -CH 2 CH 2 CH(CH 3 ) 2 , -CH 2 CH(CH 3 )(CH 2 CH 3 ), -CH
  • alkenyl refers herein to at least one point of unsaturation, ie, where two adjacent carbon atoms are connected to the alkyl group defined above by a double bond, wherein the alkyl group is as defined herein
  • alkynyl refers herein to an alkyl group in which two adjacent carbon atoms are connected by a triple bond, wherein the alkyl group is as defined herein.
  • heteroalkyl refers herein to an alkyl group that includes at least one heteroatom.
  • cycloalkyl refers herein to mono- or polycyclic carbocyclic alkyl substituents. Including cyclic alkyl, alkenyl and alkynyl. Cycloalkyl groups can be monocyclic or polycyclic (eg contain fused, bridged and/or spiro ring systems) in which the carbon atoms are located inside or outside the ring system.
  • the cycloalkyl group as a whole may have 3-14 ring atoms (for example, monocyclic cycloalkyl groups have 3-8 carbon atoms, and polycyclic cycloalkyl groups have 7-14 carbon atoms). Any suitable ring position of the cycloalkyl group can be covalently linked to the defined chemical structure.
  • cycloalkyl examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl , Norpinyl, norpinyl, adamantyl and spiro[4.5]decyl and their homologues and isomers.
  • heterocyclic or “heterocyclic” or “heterocyclyl” refers to a ring selected from the group consisting of 4-12 membered monocyclic, bicyclic and tricyclic saturated and partially unsaturated rings, except for It contains at least one carbon atom in addition to 1, 2, 3, or 4 heteroatoms from oxygen, sulfur, and nitrogen.
  • Heterocycle also refers to a 5-7 membered hetero ring fused with a 5, 6 and/or 7 membered cycloalkyl, carbocyclic aromatic or heterocyclic aromatic ring group containing at least one heteroatom selected from N, O and S Ring, provided that the point of attachment is on the heterocycle when the heterocycle is fused with a carbocyclic aromatic or heteroaromatic ring, and the point of attachment can be on the cycloalkyl or heterocycle when the heterocycle is fused with cycloalkyl.
  • Heterocycle also refers to an aliphatic spiro ring containing at least one heteroatom selected from N, O, and S, provided that the point of attachment is on the heterocycle.
  • the ring may be saturated or contain at least one double bond (ie partially unsaturated).
  • the heterocyclic ring may be substituted by oxo.
  • the point of attachment can be a carbon atom or a heteroatom in the heterocycle.
  • Heterocycle is not a heteroaryl group as defined in the text.
  • heterocycles include, but are not limited to (numbered from the preferentially designated connection position 1) 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1 -Piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperidinyl, pyranyl, 2-morpholinyl, 3-morpholinyl, ethylene oxide Oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietyl, 1,2-disulfide Heterocyclobutane, 1,3-dithiocyclobutane, dihydropyridyl, tetrahydropyridyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl , Homopiperidinyl, azepanyl (
  • Substituted heterocycles also include ring systems substituted with one or more oxo moieties, such as pyrimidinyl N-oxide, morpholinyl-N-oxidation, 1-oxo-1-thiomorpholinyl and 1, 1-dioxo-1-thiomorpholinyl.
  • oxo moieties such as pyrimidinyl N-oxide, morpholinyl-N-oxidation, 1-oxo-1-thiomorpholinyl and 1, 1-dioxo-1-thiomorpholinyl.
  • aryl refers to an aromatic ring in which each atom forming a ring is a carbon atom.
  • the aryl ring may be formed by five, six, seven, eight, nine, or more than nine carbon atoms.
  • the aryl group may be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl.
  • heteroaryl or optionally “heteroaromatic group” refers to an aromatic group which includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur.
  • the "heteroaromatic group” or “heteroaryl” portion containing N refers to an aromatic group in which at least one of the skeleton atoms of the ring is a nitrogen atom.
  • heteroaryl groups include the following:
  • the heteroaryl group may be monoradical or diradical (ie, heteroarylene group).
  • alkoxy refers to a (alkyl) O- group, where alkyl is defined herein.
  • the substituents of the compounds of the present invention are disclosed in the form of groups or ranges. This specifically means that the present invention includes each member of such groups and ranges or a sub-combination of each individual in the member.
  • C 1-6 alkyl specifically means that methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl are individually disclosed.
  • compound of the invention refers herein to compounds of formulae (I)-(III) and all pure and mixed stereoisomers, geometric isomers, tautomers , Solvates, hydrates, prodrugs and isotopically labeled compounds and any pharmaceutically acceptable salts.
  • the solvates of the compounds of the present invention refer to compounds or salts thereof combined with stoichiometric and non-stoichiometric solvents, such as hydrates, ethanolates, methanolates and the like.
  • the compound may also exist in one or more crystalline states, ie as a eutectic, polymorph, or it may exist as an amorphous solid. All such forms are covered by the claims.
  • pharmaceutically acceptable means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or the mammal being treated with it.
  • stereoisomer refers herein to compounds having one or more stereocenters with different chiralities, including the corresponding isomers and diastereomers.
  • the compounds of the present invention can be used in the form of salts, such as "pharmaceutically acceptable salts" derived from inorganic or organic acids. These include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate , Camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, glucoheptanoate, glycerol phosphate, hemisulfate, heptanoic acid Salt, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, hydrochloride, 2 -Naphthalenesulfonate, oxalate, pect
  • basic nitrogen-containing groups can be quaternized with the following reagents to form quaternary ammonium salts: such as lower alkyl halides, including methyl, ethyl, propyl and butyl chloride, bromide and iodine Compounds; such as dialkyl sulfates, including dimethyl, diethyl, dibutyl, and dipentyl sulfates; such as long-chain halides, including decyl, lauryl, myristyl, and stearyl Chloride, bromide and iodide; such as aralkyl halides, such as benzyl and phenethyl bromide.
  • lower alkyl halides including methyl, ethyl, propyl and butyl chloride, bromide and iodine Compounds
  • dialkyl sulfates including dimethyl, diethyl, dibutyl, and dipentyl sul
  • the present invention also includes isotopically-labeled compounds of the present invention, that is, the same structure as disclosed above, but one or more atoms in the structure are replaced by atoms having the same number of protons but different numbers of neutrons.
  • isotopes in combination with the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, and iodine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Cl and 131 I etc.
  • the compounds of the present invention are all within the scope of the present invention.
  • Certain isotopically-labeled compounds of the present invention such as those labeled with 3 H or 14 C, can be used in pharmaceutical tissue distribution tests. Therefore, these 3 H or 14 C isotopes are particularly preferred because of their ease of preparation and detection.
  • Isotope-labeled compounds of the formula (I)-(III) of the invention and their prodrugs can generally be prepared in this way, when performing the processes disclosed in the following procedures and/or examples and preparation examples, the isotope-labeled Reagents replace non-isotopically labeled reagents.
  • Another aspect of the present invention relates to a pharmaceutical composition containing a therapeutically effective amount of the compound of formula (I)-(III), or a pharmaceutically acceptable carrier, diluent or excipient thereof.
  • the present invention further relates to a compound described in general formulae (I)-(III) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same in preparation for the prevention and/or treatment of pathological features mediated by ALK2 kinase Use of medicines for diseases.
  • Diseases with ALK2 kinase-mediated pathological features are selected from anemia or cancer.
  • the present invention also relates to a method for treating and/or treating diseases with pathological features mediated by ALK2 kinase, which comprises administering to a patient a therapeutically effective amount of a compound of formula (I)-(III) or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, wherein the disease having ALK2 kinase-mediated pathological characteristics is selected from anemia or cancer.
  • the present invention also relates to a method for treating and/or treating diseases with pathological features mediated by ALK2 kinase, which comprises administering to a patient a therapeutically effective amount of a compound of formula (I)-(III) or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, wherein the disease having ALK2 kinase-mediated pathological characteristics is selected from anemia, wherein the anemia is selected from chronic disease anemia, chronic inflammation anemia, cancer or Anemia of progressive fibrous dysplasia.
  • the present invention also relates to a method for treating and/or treating diseases with pathological features mediated by ALK2 kinase, which comprises administering to a patient a therapeutically effective amount of a compound of formula (I)-(III) or A pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, wherein the disease having ALK2 kinase-mediated pathological characteristics is selected from cancer, wherein the cancer is selected from prostate cancer, breast cancer, cervical cancer, and uterus Endometrial cancer, colon cancer, stomach cancer, lung cancer, liver cancer, bladder cancer, pancreatic cancer, rectal cancer, skin cancer (including melanoma and basal cell carcinoma), oral cancer, bone cancer, ovarian cancer, brain cancer, head and neck cancer , Mesothelioma, leukemia, lymphoma, esophageal cancer, kidney cancer, thyroid cancer, myeloma, choriocarcinoma, testicular cancer, glioma, glioma, fallopian tube
  • the compounds provided by the present invention can be prepared by standard synthetic methods well known in the art. This specification provides general methods for preparing the compounds of the present invention.
  • the starting materials are usually commercially available, for example by Alfa Sigma- TCI, Chengdu Best Reagent, Chengdu Huana Chemical Preparation Co., Ltd., Chengdu Kelong Chemical Reagent Factory and other companies can be purchased or prepared by methods well known to those skilled in the art.
  • reaction methods and synthetic steps provide possible routes for synthesizing the compounds of the present invention and key intermediates.
  • the compounds of the present invention can also be obtained by other synthetic routes.
  • specific starting materials and reagents are used in the following reaction scheme, these starting materials and reagents can be replaced by other similar starting materials or reagents to provide various derivatives.
  • many compounds prepared by the following methods can be further modified by conventional chemical methods well known to those skilled in the art.
  • reaction starting materials, intermediates and example compounds can be separated and purified by conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography (e.g., column chromatography, TLC separation and purification, etc.).
  • reaction temperature reaction temperature
  • reaction solvent molar ratio of reactants, and/or reaction duration
  • reaction temperature reaction temperature
  • reaction solvent molar ratio of reactants, and/or reaction duration
  • the progress of the reaction can be monitored by TLC, and an appropriate time can be selected to terminate the reaction and perform post-treatment accordingly.
  • the purification conditions of the compound may also change.
  • the appropriate column chromatography eluent is selected according to the R f value of TLC, or the corresponding compound is separated and purified by preparing TLC.
  • the compound of formula (I) of the present invention can be prepared according to the following scheme 1.
  • Intermediate A and Intermediate B are acidic (trifluoroacetic acid, formic acid, acetic acid, oxalic acid, dilute hydrochloric acid, etc.) or basic (such as triethylamine, N,N-diisopropylethylamine, pyridine, 4- Under the conditions of dimethylaminopyridine, N-methylmorpholine, etc., in a suitable solvent (n-butanol, methanol, ethanol, methylene chloride, tetrahydrofuran, dioxane, dimethylformamide, 1-methyl- 2-pyrrolidone, etc.), at a suitable temperature, the reaction produces the compound of formula (I).
  • a suitable solvent n-butanol, methanol, ethanol, methylene chloride, tetrahydrofuran, dioxane, dimethylformamide, 1-methyl- 2-pyrrolidone, etc
  • reaction conditions are not limiting, and the method described can be used to prepare compounds of formula (I) by reasonably changing the reaction conditions.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , m and X have the definitions as described in the present invention, and L is a leaving group.
  • N-(2-chloropyrimidin-4-yl)-2,2'-bipyridin-3-amine 100mg, 0.35mmol
  • 8-methoxy-2,3-di Hydrobenzo[b][1,4]dioxan-6-amine 96mg, 0.53mmol
  • TFA 157uL, 2.11mmol
  • 3mL n-BuOH under argon protection at 85 °C for 1h.
  • Promega company ALK2Kinase Enzyme System kit was used for detection. First prepare the reagents for kinase detection, including: 1 4x Kinase buffer 250 ⁇ L (including 200 ⁇ M DTT); 2 5x Inhibitor mother liquid, that is, the test compound is prepared with DMSO into 10mM mother liquid, and then the test compound is diluted with DMSO to 5mM (20 ⁇ L 10mM test compound) Stock solution + 20 ⁇ L DMSO dissolved); 3 1x Kinase buffer (containing 1% DMSO) 200 ⁇ L; 4 1x Kinase buffer 40 ⁇ L; 5 Kinase solution 115 ⁇ L; 6 Substrate Mix (containing 65 ⁇ M ATP) 120 ⁇ L.
  • 1 4x Kinase buffer 250 ⁇ L including 200 ⁇ M DTT
  • 2 5x Inhibitor mother liquid that is, the test compound is prepared with DMSO into 10mM mother liquid, and then the test compound is diluted with DMSO to 5mM (20 ⁇ L 10
  • inhibitor working solution that is, test compound is prepared into 5mM mother solution with DMSO, take 1 ⁇ L of 5mM mother solution and add 99 ⁇ L 1xKinase buffer to dilute to 50000nM concentration; then take 20 ⁇ L of this concentration solution and add 80 ⁇ L 1xKinase buffer (containing 1% DMSO) to obtain 10000nM
  • concentration is diluted 5 times in this way, and a total of 6 concentration gradients are set.
  • the IC 50 value ranges indicated by A, B, and C are: A ⁇ 150nM, 150nM ⁇ B ⁇ 500nM, and C ⁇ 500nM.
  • test results show that the compounds of the examples of the present invention have a significant inhibitory effect on ALK2 kinase.
  • Digest HepG2 cells (ATCC HB-8065) in the exponential growth phase with trypsin-EDTA, inoculate 5000 cells/well into 96-well plates, and culture in MEM (10% FBS) medium at 37°C under 5% CO2 After 24 hours, each culture well was added with a test compound dissolved in DMSO and diluted with a medium at a gradient.
  • the preparation method of the test compound is to first prepare the mother liquor of the compound at a concentration of 10 mM, and then to dilute the compound in a gradient, the highest concentration of the compound to be detected is 10 ⁇ M, and the dilution is 5 times, a total of 8 gradients. Add the diluted compound to the 96-well plate with cells, 50 ⁇ L per well.
  • hepcidin ELISA kit Human Hepcidin Quantikine ELISA Kit, R&D systems, Cat#DHP250 was used to quantitatively detect hepcidin protein.
  • Preparation reagents include: Calibrator, Diluent, RD5-26, 80mL, Wash, Buffer, 500mL, Substrate, Solution, 30mL (15mL of chromogen A and 15mL of B, etc., mix well, use in the dark for 15 minutes, the final volume of each well is 200 ⁇ L), Human Hepcidin standard: The final concentration of recombinant human Hepcidin standard is 1000, 500, 250, 125, 62.5, 31.3, 15.6, 0 (pg/mL). Add 50 ⁇ L of Assay Diluent RD1-21 to each well. Add 50 ⁇ L of standard, control or sample to each well, cover with a sealing strip, and incubate at room temperature for 2 hours.
  • IC 50 values indicated by A, B, and C are: A ⁇ 200nM, 200nM ⁇ B ⁇ 500nM, and C ⁇ 500nM.
  • test results show that the compounds of the examples of the present invention have a significant inhibitory effect on Hepcidin expression in HepG2 cells.

Abstract

本发明涉及一类2,4-二氨基嘧啶衍生物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，及其制备方法以及该类化合物单独或与其他药物联合使用在治疗具有ALK2激酶介导的病理学特征的疾病中的应用。

Description

2,4-二氨基嘧啶衍生物 技术领域
本发明涉及一类2,4-二氨基嘧啶衍生物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，及其制备方法以及该类化合物单独或与其他药物联合使用在治疗具有ALK2激酶介导的病理学特征的疾病中的应用。
背景技术
全身铁平衡是通过协调调节十二指肠铁吸收，巨噬细胞中衰老红细胞的铁再循环，以及肝脏中储存的铁的调动来维持的(Ganz T.,Physiol Rev.2013；93(4):1721–1741)。这个过程中的一个关键协调器是主要在肝细胞中合成的一种小肽类激素——铁调素(Hepcidin)(Krause A.等,FEBS Lett.2000；480(2–3):147–150；Pigeon C.等,J Biol Chem.2001；276(11):7811–7819；Park CH.等,J Biol Chem.2001；276(11):7806–7810)。Hepcidin通过结合并诱导膜铁运输蛋白(FPN1)的内化和降解，减少了十二指肠铁的吸收和铁从单核细胞/巨噬细胞的输出(Hentze MW.等,Cell.2010；142(1):24–38；Nemeth E.等,Science.2004；306(5704):2090–2093；Theurl I.等,Blood.2009；113(21):5277–5286)。因此，血清hepcidin水平升高增强了铁在网状内皮***中的储存并导致可用的铁降低和缺铁性性红细胞的生成。Hepcidin表达异常升高会引起人体严重的功能性缺铁性贫血，并且是慢性病贫血(ACD)的病理生理学的核心(Weiss G.等,N Engl J Med.2005；352(10):1011–1023)。Hepcidin在肝脏的转录主要受几个因素的影响，包括机体铁的储存，红细胞生成对铁的需求，缺氧和炎症反应。骨形成蛋白(BMPs)在介导这些因素和通过激活BMP受体(BMPR)-SMAD信号传导驱动铁调素转录诱导中发挥重要作用(Wang RH.等,Cell Metab.2005；2(6):399–409；Andriopoulos B Jr.等,Nat Genet.2009；41(4):482–487；Meynard D.等,Nat Genet.2009；41(4):478–481；Zhao N.等,J Clin Invest.2013；123(6):2337–2343)。BMPR激活素受体样激酶2(ALK2)和ALK3在这一过程中已经显示出重要作用，肝脏特异性缺失ALK2或ALK3阻断BMP配体结合下游的hepcidin生成的诱导并导致小鼠中的铁超载(Steinbicker AU.等,Blood.2011；118(15):4224–4230)。
BMP配体与骨形成蛋白受体(BMPRs)相互作用，BNPRs属于TGF-β超家族的丝氨酸/苏胺酸激酶受体。慢性感染、炎症及恶性肿瘤所致贫血通常称为慢性病贫血，在这些患者中，BMP信号通过两个BMP I型受体，ALK2和ALK3诱导hepcidin的肝表达。Hepcidin通过降解铁输出，铁转运蛋白减少血液铁水平，导致巨噬细胞和其他细胞中的铁储存的增加，并使其不能用于血红蛋白和红细胞功能。增加铁摄入不能逆转慢性病贫血(ACD)，因为由于BMP通路的激活和高血浆hepcidin水平摄取的铁只是被储存。阻断BMP信号传导是治疗慢性病贫血的有潜力的疗法。
BMP信号通路还在肿瘤细胞的生长和恶化中发挥重要作用，特别是乳腺癌、***癌，和其他容易发 生骨转移的肿瘤(Lin Ye.等，Front Biosci.2011(16):865-897)。BMPs和BMPRs在转移乳腺癌中的表达高于没有转移的，也在骨转移的***癌中高表达。这些结果表明BMP抑制剂可能可以阻止骨转移。
发明内容
本发明的目的在于提供一种式(I)所示的化合物或其药学上可接受的盐、水合物、溶剂化物或立体异构体，
Figure PCTCN2019128246-appb-000001
其中：
X为-(CR 7R 8) p-；
R 1选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基，其中烷基、烯基、炔基、杂烷基、环烷基、杂环基、芳基或杂芳基可被任意取代；
R 2选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9或-NR 9COR 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 3、R 4和R 5分别独立地选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9、-SO 2R 9、-NR 9COR 10、-NR 9CONR 10R 11、-NR 9CO 2R 10、-NR 9SO 2NR 10R 11或-NR 9SO 2R 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 6选自-H、卤素、-OH、-CN、-CF 3、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基或杂烷基，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基、杂烷基、-C 2-6烯基、-C 2-6炔基、饱和或不饱和的杂环基、氧代或3-8元环烷基；其中烷基、烯基、炔基、杂环基或环烷基可被任意取代；
R 9、R 10和R 11分别独立地选自-H、杂烷基、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基；其中(R 9和R 10)和/或(R 10和R 11)连同与它们相连的氮原子一起形成可被至少一个R 12取代的饱和或不饱和的杂环；
每个R 12分别独立地选自-H、卤素、-C 1-6烷基或氧代；
m为0，1，2或3；
p为1，2或3；
q为0，1，2或3。
在另一个方面，本发明提供给式(II)化合物，
Figure PCTCN2019128246-appb-000002
其中，
X为-(CR 7R 8) p-；
R 1选自可被任意取代的芳基或杂芳基；
R 2选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9或-NR 9COR 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 3、R 4和R 5分别独立地选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9、-SO 2R 9、-NR 9COR 10、-NR 9CONR 10R 11、-NR 9CO 2R 10、-NR 9SO 2NR 10R 11或-NR 9SO 2R 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 6选自-H、卤素、-OH、-CN、-CF 3、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基或杂烷基，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基、杂烷基、-C 2-6烯基、-C 2-6炔基、饱和或不饱和的杂环基、氧代或3-8元环烷基；其中烷基、烯基、炔基、杂环基或环烷基可被任意取代；
R 9、R 10和R 11分别独立地选自-H、杂烷基、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基；其中(R 9和R 10)和/或(R 10和R 11)连同与它们相连的氮原子一起形成可被至少一个R 12取代的饱和或不饱和的杂环；
每个R 12分别独立地选自-H、卤素、-C 1-6烷基或氧代；
p为1，2或3；
q为0，1，2或3。
在另一个方面，本发明提供给式(III)化合物，
Figure PCTCN2019128246-appb-000003
其中，
X为-(CR 7R 8) p-；
R 1选自可被任意取代的杂芳基；
R 2选自-H、卤素或-C 1-6烷基；其中烷基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
R 3和R 4分别独立地选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、C 1-6烷基、C 2-6烯基、C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9、-SO 2R 9、-NR 9COR 10、-NR 9CONR 10R 11、-NR 9CO 2R 10、-NR 9SO 2NR 10R 11或-NR 9SO 2R 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
R 6选自-H、卤素、-OH、-CN、-CF 3、-NO 2、-C 1-6烷基或杂烷基，其中烷基或杂烷基可被任意取代；
R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基、杂烷基、-C 2-6烯基、-C 2-6炔基或氧代；其中烷基、烯基或炔基可被选自-H、卤素、-C 1-4烷基的取代基取代；
R 9、R 10和R 11分别独立地选自-H、杂烷基、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基；其中(R 9和R 10)和/或(R 10和R 11)连同与它们相连的氮原子一起形成可被至少一个R 12取代的饱和或不饱和的杂环；
每个R 12分别独立地选自-H、卤素、-C 1-6烷基或氧代；
p为1，2或3；
q为0，1，2或3。
在进一步的实施方式中，本发明提供式(III)化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
X为-(CR 7R 8) p-；
R 1选自可被任意取代的吡啶基；
R 2选自-H、卤素或-C 1-6烷基；其中烷基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
R 3和R 4分别独立地选自-H、卤素、-C 1-6烷基或杂烷基；其中烷基或杂烷基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
R 6选自-H、卤素、-OH、-CN、-C 1-6烷基或-C 1-6烷氧基；其中烷基或烷氧基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基或-C 1-6烷氧基，其中烷基或烷氧基可被选自-H、卤素或-C 1-4烷基的取代基取代；
p为1或2。
在进一步的实施方式中，本发明提供式(III)化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
X为-(CR 7R 8) p-；
R 1为
Figure PCTCN2019128246-appb-000004
其中R 13选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-NO 2、-NH 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基或杂烷基；
R 2选自-H、卤素或C 1-6烷基；
R 3和R 4分别独立地选自-H、卤素、C 1-6烷基或-C 1-6烷氧基，其中烷基或烷氧基可被选自-H、卤素或-C 1-4烷基或氧代的取代基取代；
R 6选自-H、卤素、-OH、-CN、-C 1-6烷基或-C 1-6烷氧基；
R 7和R 8分别独立地选自-H、卤素或-C 1-6烷基，其中烷基可被选自-H、卤素或-C 1-4烷基的取代基取代；
p为1或2。
在进一步的实施方式中，本发明提供式(III)化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
X为-(CR 7R 8) p-；
R 1为
Figure PCTCN2019128246-appb-000005
其中R 13选自-H、卤素或-C 1-4烷基；
R 2选自-H或-C 1-4烷基；
R 3选自-H、-C 1-4烷基或-C 1-4烷氧基；
R 4选自-H或-C 1-4烷基；
R 6选自-H、卤素、-CN、-C 1-4烷基或-C 1-4烷氧基；
R 7和R 8分别独立地选自-H或-C 1-4烷基；
p为1或2。
本发明涉及式(I)-式(III)的典型化合物如下，但并不限于以下化合物：
Figure PCTCN2019128246-appb-000006
Figure PCTCN2019128246-appb-000007
定义
如上文和本文其它地方所用，下列术语和缩写具有下面所定义的含义。如未定义，则本说明书所使用的所有技术和科学术语均具有本领域普通技术人员通常所理解的含义。
缩写 含义
ALK2 激活素受体样激酶2
Hepcidin 铁调素
FPN1 膜铁运输蛋白
ACD 慢性病贫血
BMPs 骨形成蛋白
BMPR 骨形成蛋白受体
TGF-β 转化生长因子-β
TLC 薄层色谱
R f 比移值
n-BuOH 正丁醇
DCM 二氯甲烷
TFA 三氟醋酸
EA 乙酸乙酯
ADP 二磷酸腺苷
ATP 三磷酸腺苷
Ser/Thr 丝氨酸/苏氨酸
术语“卤素”在本文中是指-F、-Cl、-Br和-I。
术语“烷基”在本文中是指不含有杂原子的烷基。因此，该术语包括直链烷基如甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基、十一烷基、十二烷基等。该术语还包括支链烷基的支链异构体，包括但不限于例如下面的基团：-CH(CH 3) 2、-CH(CH 3)(CH 2CH 3)、-CH(CH 2CH 3) 2、-C(CH 3) 3、 -C(CH 2CH 3) 3、-CH 2CH(CH 3) 2、-CH 2CH(CH 3)(CH 2CH 3)、-CH 2CH(CH 2CH 3) 2、-CH 2C(CH 3) 3、-CH 2C(CH 2CH 3) 3、-CH(CH 3)-CH(CH 3)(CH 2CH 3)、-CH 2CH 2CH(CH 3) 2、-CH 2CH 2CH(CH 3)(CH 2CH 3)、-CH 2CH 2CH(CH 2CH 3) 2、-CH 2CH 2C(CH 3) 3、-CH 2CH 2C(CH 2CH 3) 3、-CH(CH 3)CH 2CH(CH 3) 2、-CH(CH 3)CH(CH 3)CH(CH 3) 2、-CH(CH 2CH 3)CH(CH 3)CH(CH 3)(CH 2CH 3)等。因此术语烷基包括伯烷基、仲烷基和叔烷基。
术语“烯基”在本文中是指其中有至少一个不饱和点，即，其中两个相邻碳原子通过双键连接上面所定义的烷基，其中所述烷基如本文中所定义
术语“炔基”在本文中是指涉及其中两个相邻碳原子通过三键连接的烷基，其中所述烷基如本文中所定义。
术语“杂烷基”在本文中是指包括至少一个杂原子的烷基。
术语“环烷基”在本文中是指单-或多环的碳环烷基取代基。包括环状烷基、烯基和炔基。环烷基可以使单环或多环(例如含有稠合、桥连和/或螺环系)，其中碳原子位于环系内侧或外侧。环烷基作为整体可以具有3-14个环原子(例如就单环环烷基而言具有3-8个碳原子，就多环环烷基而言具有7-14个碳原子)。环烷基的任意适当的环位置可以与所定义的化学结构共价连接。环烷基的实例包括环丙基、环丁基、环戊基、环己基、环庚基、环戊烯基、环己烯基、环己二烯基、环庚三烯基、降冰片基、降蒎烷基、降蒈烷基、金刚烷基和螺[4.5]癸基及其同系物和异构体等。
术语“杂环的”或“杂环”或“杂环基”指的是选自如下的环：4-12元的单环、双环和三环的饱和和部分不饱和的环，其除了选自氧、硫和氮的1、2、3或者4个杂原子外包含至少一个碳原子。“杂环”也指与5、6和/或7元环烷基、碳环芳族或杂环芳香环族稠合并包含至少一个选自N、O和S的杂原子的5-7元杂环，条件是当杂环与碳环芳香族或杂芳族环稠合时连接点在杂环上，且当杂环与环烷基稠合时连接点可在环烷基或杂环上。
“杂环”也指包含至少一个选自N、O和S的杂原子的脂族螺环，条件是连接点在杂环上。所述环可以是饱和的或含有至少一个双键(即部分不饱和的)。所述杂环可被氧代(oxo)取代。连接点可以是杂环中的碳原子或杂原子。杂环不是文本中定义的杂芳基。
杂环的实例包括，但不局限于(从优先指定的连接位置1编号)1-吡咯烷基、2-吡咯烷基、2,4-咪唑烷基、2,3-吡唑烷基、1-哌啶基、2-哌啶基、3-哌啶基、4哌啶基、2,5-哌啶基、吡喃基、2-吗啉基、3-吗啉基、环氧乙烷基(oxiranyl)、氮杂环丙烯基(aziridinyl)、环硫乙烷基(thiiranyl)、氮杂环丁烷基、氧杂环丁烷基、硫杂环丁烷基、1,2-二硫杂环丁烷基、1,3-二硫杂环丁烷基、二氢吡啶基、四氢吡啶基、硫代吗啉基、噻噁烷基(thioxanyl)、哌嗪基、高哌嗪基、高哌啶基、氮杂环庚烷基(azepanyl)、氧杂环庚烷基(oxepanyl)、硫杂环庚烷基(thiepanyl)、1，4-氧硫杂环己烷基、1,4-二氧杂环庚烷基、1,4-氧硫杂环庚烷基、1,4-氧氮杂环庚烷基、1,4-二硫杂环庚烷基、1,4-硫氮杂环庚烷基和1,4-二氮杂环庚烷1,4-二噻烷基、1,4-氮硫杂环己 烷基、氧氮杂卓基、二氮杂卓基、硫氮杂卓基、二氢噻吩基、二氢吡喃基、二氢呋喃基、四氢呋喃基、四氢噻吩基、四氢吡喃基、四氢噻喃基、1-吡咯啉基、2-吡咯啉基、3-吡咯啉基、吲哚啉基、2H-吡喃基、4H-吡喃基、1,4-二噁烷基、1,3-二氧环戊基、吡唑啉基、吡唑烷基、二硫杂环己烷基、二硫环戊基、吡唑烷基咪唑烷基(pyrazolidinylimidazolinyl)、嘧啶酮基、1,1-二氧代-硫代吗啉基、3-氮杂双环[3.1.0]己烷基、3-氮杂双环[4.1.0]庚烷基、氮杂双环[2.2.2]己烷基。取代的杂环还包括被一个或多个氧代部分取代的环体系，如嘧啶基N-氧化物、吗啉基-N-氧化、1-氧代-1-硫代吗啉基和1,1-二氧代-1-硫代吗啉基。
术语“芳基”是指其中形成环的每个原子是碳原子的芳香族环。芳基环可以由五个、六个、七个、八个、九个或超过九个碳原子形成。芳基基团可以是任选取代的。芳基基团的实例包括但不限于苯基、萘基、菲基、蒽基、芴基和茚基。
术语“杂芳基”或选择性的“杂芳香族基”是指芳香族基团，其中包括自选氮，氧和硫的一个或更多个环杂原子。含有N的“杂芳香族基”或“杂芳基”部分是指芳香族基团，其中环的骨架原子至少一个是氮原子。杂芳基基团的说明性实例包括以下几个部分：
Figure PCTCN2019128246-appb-000008
等。根据结构，杂芳基基团可以是单自由基或双自由基的(即，杂亚芳基基团)。
术语“氧代”在本文中是指(＝O)或(-O -)。
术语“烷氧基”是指(烷基)O-基团，其中烷基是在此定义的。
在本说明书的各个位置，本发明化合物的取代基以基团或范围的形式进行公开。这具体意味着本发明包括这样的基团和范围的每个成员或成员中的每个个体的亚组合。如术语“C 1-6烷基”具体意味着单独公开了甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
术语“本发明化合物”(除非另有具体指明)在本文中是指式(I)-(III)化合物及其所有纯的和混合的立体异构体、几何异构体、互变异构体、溶剂合物、水合物、前药及同位素标记的化合物和任何药学上可接受的盐。本发明化合物的溶剂合物是指与化学计量和非化学计量的溶剂结合的化合物或其盐，如水合物、乙醇合物、甲醇合物等。化合物也可以一种或多种结晶状态存在，即作为共晶体、多晶型物，或其可以无定形固体存在。所有此种形式均被权利要求所涵盖。
术语“药学上可接受”表示物质或组合物在化学上和/或毒理学上必须与构成制剂的其它成分和/或用其治疗的哺乳动物相容。
术语“立体异构体”在本文中是指具有一个或多个立体中心的手性不同的化合物，包括对应异构体和非对映异构体。
本发明化合物可以以盐的形式被使用，如从无机酸或有机酸衍生得到的“医药上可接受的盐”。这些包括但并不限于下列所述：乙酸盐、己二酸盐、藻酸盐、柠檬酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、丁酸盐、樟脑酸盐、樟脑磺酸盐、二葡萄糖酸盐、环戊烷丙酸盐、十二烷基硫酸盐、乙磺酸盐、葡糖庚酸盐、甘油磷酸盐、半硫酸盐、庚酸盐、己酸盐、延胡索酸盐、氢氯化物、氢溴酸盐、氢碘酸盐、2-羟基乙磺酸盐、乳酸盐、马来酸盐、甲磺酸盐、盐酸盐、2-萘磺酸盐、草酸盐、果胶酯酸盐、硫酸盐、3-苯基丙酸盐、苦味酸盐、三甲基乙酸盐、丙酸盐、琥珀酸盐、酒石酸盐、硫氰酸盐、对甲苯磺酸盐和癸酸盐。另外，碱性含氮基团可以与以下试剂发生季铵化反应生成季铵盐：如低碳烷基卤化物，包括甲基、乙基、丙基和丁基的氯化物、溴化物和碘化物；如二烷基硫酸盐，包括二甲基、二乙基、二丁基和二戊基的硫酸盐；如长链卤化物，包括癸基、月桂基、肉豆蔻基和硬脂基的氯化物、溴化物和碘化物；如芳烷基卤化物，如苯甲基和苯乙基的溴化物等。
本发明还包括同位素标记的本发明化合物，即与上述所公开的结构相同，但该结构中一个或多个原子被与其具有相同质子数但不同中子数的原子所替代。结合本发明化合物的同位素实施例包括氢、碳、氮、氧、硫、氟、氯、碘的同位素，分别如 2H、 3H、 13C、 14C、 15N、 18O、 17O、 35S、 18F、 36Cl和 131I等。本发明的化合物，其立体异构体、互变异构体或医药上可接受的盐，以及含有上述同位素和/或其他原子同位素的所述以上形式的化合物，均在本发明范围内。某些同位素标记的本发明化合物，如被 3H或 14C所标记的那些化合物可以用于药物组织分布试验中，因此，这些 3H或 14C同位素由于其容易制备和检测是特别优选的。此外，被较重的同位素如 2H所替代的某些本发明化合物由于具有更好的代谢稳定性而具有某些治疗优势，如可以增加体内半衰期和较少剂量等，因此， 2H在某些情况下也是优选的。同位素标记的本发明式(I)-(III)化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。
本发明的另一方面涉及一种药物组合物，其含有治疗有效量的通式(I)-(III)所述的化合物，或其药学上可接受的载体、稀释剂或赋形剂。
本发明进一步涉及通式(I)-(III)所述的化合物或其可药用的盐，或包含其的药物组合物在制备用于预防和/或治疗具有ALK2激酶介导的病理学特征的疾病的药物中的用途。具有ALK2激酶介导的病理学特征的疾病选自贫血或癌症。
本发明还涉及一种治疗预防和/或治疗预防具有ALK2激酶介导的病理学特征的疾病的方法，其包括向 患者施用治疗有效量的通式(I)-(III)所述的化合物或其可药用的盐，或包含其的药物组合物，其中所述具有ALK2激酶介导的病理学特征的疾病选自贫血或癌症。
本发明还涉及一种治疗预防和/或治疗预防具有ALK2激酶介导的病理学特征的疾病的方法，其包括向患者施用治疗有效量的通式(I)-(III)所述的化合物或其可药用的盐，或包含其的药物组合物，其中所述具有ALK2激酶介导的病理学特征的疾病选自贫血，其中所述的贫血选自慢性病性贫血、慢性炎症贫血、癌症或进行性纤维发育不良贫血。
本发明还涉及一种治疗预防和/或治疗预防具有ALK2激酶介导的病理学特征的疾病的方法，其包括向患者施用治疗有效量的通式(I)-(III)所述的化合物或其可药用的盐，或包含其的药物组合物，其中所述具有ALK2激酶介导的病理学特征的疾病选自癌症，其中所述的癌症选自***癌、乳腺癌、***、子宫内膜癌、结肠癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、直肠癌、皮肤癌(包括黑色素瘤和基底细胞癌)、口腔癌、骨癌、卵巢癌、脑癌、头颈部癌、间皮内膜癌、白血病、淋巴瘤、食管癌、肾癌、甲状腺癌、骨髓瘤、绒毛膜癌、睾丸癌、神经胶质瘤、母神经胶质瘤、输卵管肿瘤、骨髓纤维化、真性红细胞增多症或特发性血小板增多症。
化合物或中间体制备方法
为描述本发明，以下列出了具体实施例。但需要理解，本发明不限于这些实施例，以下实施例只是提供实践本发明的方法，并不以任何方式限制本发明的范畴。
本发明提供的化合物可以通过本领域公知的标准合成方法来制备，本说明书提供了制备本发明化合物的一般方法。起始原料通常可通过商业化获得，例如通过Alfa
Figure PCTCN2019128246-appb-000009
Sigma-
Figure PCTCN2019128246-appb-000010
TCI、成都贝斯特试剂、成都华娜化学制剂有限公司、成都科龙化工试剂厂等公司购买得到，或者通过本领域技术人员所熟知的方法进行制备。
下述反应方法及合成步骤提供了用于合成本发明化合物以及关键中间体的可能途径。关于个别反应步骤的更详细说明，参见下述实施例。本领域技术人员应理解，本发明化合物也可以通过其它的合成途径获得。虽然下文反应流程中使用了特定的起始原料和试剂，但是这些起始原料和试剂可以被其它类似的起始原料或试剂所取代，以提供各种衍生物。此外，在本说明书的指导下，通过下述方法制得的许多化合物可以通过本领域技术人员所熟知的常规化学方法进行进一步修饰。
下文通过实施例与制备进一步解释并列举本发明化合物及相应的制备方法。应了解，尽管具体实施例中给出了典型或优选的反应条件(如反应温度、时间、反应物的摩尔比、反应溶剂以及压力等)，但是本领域技术人员也可以使用其它反应条件。最佳反应条件可随所用的特定反应底物或溶剂而发生改变，但所述条件可由所属领域的技术人员通过常规优化而确定。
反应起始原料、中间体以及实施例化合物可以通过沉淀、过滤、结晶、蒸发、蒸馏以及色谱法(如柱 层析法、TLC分离纯化等)等常规技术进行分离与纯化。
试验中使用的商品化溶剂及试剂如无特殊说明，购买后均无需进一步纯化或处理直接使用。参考其它实施例或合成方法时，反应条件(反应温度、反应溶剂、反应物摩尔比或/和反应持续时间)可能不同。一般而言，可通过TLC监测反应进程，据此选择合适的时间终止反应并进行后处理。化合物的纯化条件也可能发生变化，一般而言，依据TLC的R f值选择合适的柱层析洗脱剂，或通过制备TLC分离纯化相应化合物。
本发明式(I)化合物可以按照以下流程1加以制备。中间体A和中间体B在酸性(三氟醋酸、甲酸、乙酸、乙二酸、稀盐酸等)或者碱性(如三乙胺、N,N-二异丙基乙胺、吡啶、4-二甲氨基吡啶、N-甲基吗啉等)条件下，在合适的溶剂(正丁醇、甲醇、乙醇、二氯甲烷、四氢呋喃、二噁烷、二甲基甲酰胺、1-甲基-2-吡咯烷酮等)中，在合适的温度下，反应生成式(I)化合物。应了解，这些反应条件并不是限制性的，所述的方法能够通过合理变化反应条件用于制备式(I)化合物。其中R 1、R 2、R 3、R 4、R 5、R 6、m和X具有如本发明所述的定义，L为离去基团。
流程1
Figure PCTCN2019128246-appb-000011
下列实施例打算阐述特定的发明实施方式，决不打算限制本发明说明书或权利要求书的范围。本领域技术人员将认可的是，原料可以不同，可以采用额外步骤来生成本发明涵盖的化合物，正如下列实施例所证明的。下列实施例仅供阐述目的，既不打算、也不应当以任意方式被解释为限制发明。本领域技术人员将认识到的是，可以进行变化和修改，而不违背本发明的精神或范围。
实施例1、制备N 4-(2,2'-联吡啶-3-基)-N 2-(7-甲氧基苯并[d][1,3]二氧杂环戊烯-5-基)嘧啶-2,4-二胺(化 合物1)
Figure PCTCN2019128246-appb-000012
Figure PCTCN2019128246-appb-000013
在圆底烧瓶中，依次加入N-(2-氯嘧啶-4-基)-2,2’-联吡啶-3-胺(100mg，0.35mmol)(制备方法参考WO2014151871 A2)、7-甲氧基苯并[d][1,3]二氧杂环戊烯-5-胺(88.4mg，0.53mmol)(制备方法参考J.Chin.Chem.Soc.,2001,48,59-63)、TFA(157uL，2.11mmol)和5mL n-BuOH，氩气保护下85℃反应1.5h。反应结束，冷至室温，加入饱和碳酸氢钠溶液，DCM(20mL×3)萃取，有机层用无水硫酸钠干燥，移除溶剂得粗产品；粗产品分别通过DCM和EA打浆纯化得到化合物1(122mg，灰白色固体，产率84.2％)。 1H NMR(DMSO-d 6,400MHz,ppm):δ＝12.86(s,1H),9.22-9.18(m,2H),8.79-8.78(m,1H),8.58-8.56(d,J＝8.2Hz,1H),8.37-8.35(dd,J＝4.4,1.4Hz,1H),8.13-8.12(d,J＝5.7Hz,1H),8.08-8.04(m,1H),7.56-7.52(m,1H),7.43-7.40(m,1H),7.17(s,1H),6.96(s,1H),6.48-6.46(d,J＝5.7Hz,1H),5.95(s,2H),3.79(s,3H).ESI-MS m/z：415.4[M+H] +.
实施例2、制备N 4-(2,2'-联吡啶-3-基)-N 2-(8-甲氧基-2,3-二氢苯并[b][1,4]二氧杂环己烯-6-基)嘧啶-2,4- 二胺(化合物2)
Figure PCTCN2019128246-appb-000014
制备N 4-(2,2'-联吡啶-3-基)-N 2-(8-甲氧基-2,3-二氢苯并[b][1,4]二氧杂环己烯-6-基)嘧啶-2,4-二胺(化合 物2)
Figure PCTCN2019128246-appb-000015
在圆底烧瓶中，依次加入N-(2-氯嘧啶-4-基)-2,2’-联吡啶-3-胺(100mg，0.35mmol)、8-甲氧基-2,3-二氢苯并[b][1,4]二氧杂环己烯-6-胺(96mg，0.53mmol)(制备方法参考J.Chin.Chem.Soc.,2001,48,59-63和J.Med.Chem.2004,47,3823-3842)、TFA(157uL，2.11mmol)和3mL n-BuOH，氩气保护下85℃反应1h。反应结束，冷至室温，加入水，DCM(20mL×3)萃取，有机层用无水硫酸钠干燥，移除溶剂，通过硅胶柱(DCM:MeOH＝50：1)纯化得到化合物2(115mg，白色固体，产率76.7％)。 1H NMR(CDCl 3,400MHz,ppm):δ＝12.87(s,1H),9.12-9.09(dd,J＝8.5,1.2Hz,1H),8.64-8.61(m,2H),8.32-8.30(dd,J＝4.4,1.3Hz,1H),8.09-8.07(d,J＝5.7Hz,1H),7.91-7.85(m,1H),7.35-7.31(m,1H),7.28-7.25(m,2H),6.84-6.83(d,J＝2.3Hz,1H),6.75-6.74(d,J＝2.3Hz,1H),6.24-6.22(d,J＝5.7Hz,1H),4.34-4.29(m,4H),3.81(s,3H). 13C NMR(CDCl 3,100MHz,ppm):δ＝160.6,160.1,158.4,156.6,148.7,146.3,143.9,141.7,139.6,137.4,137.3,132.7,129.1,128.8,124.1,123.4,122.9,102.8,100.5,98.5,64.5,56.2.ESI MS(M+H) +＝429.4.
实施例3、制备N 2-(7-甲氧基苯并[d][1,3]二氧杂环戊烯-5-基)-N 4–(6-甲基-2,2'-联吡啶-3-基)嘧啶-2,4- 二胺(化合物3)
Figure PCTCN2019128246-appb-000016
参照流程1的制备方案，得到化合物3，ESI-MS：429.4[M+H] +。
实施例4、制备N 4-(2,2'-联吡啶-3-基)-5-氯-N 2-(7-甲氧基苯并[d][1,3]二氧杂环戊烯-5-基)嘧啶-2,4-二胺 (化合物4)
Figure PCTCN2019128246-appb-000017
参照流程1的制备方案，得到化合物4，ESI-MS：449.3[M+H] +。
实施例5、制备N 4-(2,2'-联吡啶-3-基)-5-甲氧基-N 2-(8-甲氧基-2,3-二氢苯并[b][1,4]二氧杂环己烯-6-基) 嘧啶-2,4-二胺(化合物5)
Figure PCTCN2019128246-appb-000018
参照流程1的制备方案，得到化合物5，ESI-MS：459.3[M+H] +。
实施例6、制备N 4-(2,2'-联吡啶-3-基)-N 2-(7-甲氧基-2,2-二甲基苯并[d][1,3]二氧杂环戊烯-5-基)嘧啶-2,4- 二胺(化合物6)
Figure PCTCN2019128246-appb-000019
参照流程1的制备方案，得到化合物6，ESI-MS：443.2[M+H] +。
生物测试
1.化合物对ALK2的抑制活性试验
采用Promega公司ALK2Kinase Enzyme System kit进行检测。首先配制激酶检测所用试剂，包括：①4x Kinase buffer 250μL(含200μM DTT)；②5x Inhibitor母液，即将受试化合物用DMSO配制成10mM母液，再用DMSO将受试化合物稀释成5mM(20μL 10mM受试化合物母液+20μL DMSO溶解)；③1xKinase  buffer(含1％DMSO)200μL；④1x Kinase buffer 40μL；⑤Kinase solution 115μL；⑥Substrate Mix(含65μM ATP)120μL。然后配制抑制剂工作液，即将受试化合物用DMSO配制成5mM母液，取1μL 5mM母液加入99μL 1xKinase buffer稀释至50000nM浓度；随后取该浓度溶液20μL加入80μL 1xKinase buffer(含1％DMSO)，获得10000nM浓度，以此方法进行5倍稀释，共设6个浓度梯度。每个浓度的受试化合物取1μL加入至384孔板中，随后每孔分别加入2μL Kinase solution(160mM Tris,7.5；80mM MgCl 2；0.4mg/ml BSA；200μM DTT；ALK2激酶)，室温下1000rpm离心20s，于25℃孵育30min；随后每孔加入2μL含ATP和激酶底物的混合液，室温下1000rpm离心20s，于25℃孵育120min。随后每孔加入5μL的ADP-Glo TM试剂，于25℃孵育40min；最后每孔加入10μL激酶检测液(Kinase Detection Reagent)，于25℃孵育30min后检测；用酶标仪记录化学发光值(luminescence)，用Origin 7.5拟合曲线，计算受试物对ALK2激酶活性的IC 50(nM)值。
表1.化合物对ALK2的抑制活性测试结果。
Figure PCTCN2019128246-appb-000020
*化合物I为依照WO2014151871 A2制备的专利中化合物12。
表1中，A、B、C表示的IC 50值范围分别为：A≤150nM，150nM<B<500nM，C≥500nM。
测试结果表明本发明实施例化合物对ALK2激酶具有显著的抑制作用。
2.化合物对HepG2细胞中Hepcidin(铁调素)表达的抑制试验
将处于指数生长期的HepG2细胞(ATCC HB-8065)用胰酶-EDTA消化，以5000个细胞/孔接种至96孔板，于37℃5％CO2条件下MEM(10％FBS)培养基培养24小时后，各培养孔加入溶于DMSO，培养基稀释的梯度浓度的受试化合物。受试化合物配制方法为首先配制成10mM浓度的化合物母液，然后将化合物梯度稀释，化合物检测最高浓度为10μM，5倍稀释共8个梯度。将稀释好的化合物加入到铺有细胞的96孔板中，每孔50μL。加药后放入37℃5％CO 2的孵箱中，再孵育48小时后，孵育结束后，将96孔板室温下离心1000rpm×5min，取上清，于30min内完成ELISA样品加样和检测。以铁调素ELISA试剂盒(Human Hepcidin Quantikine ELISA Kit，R&D systems，Cat#DHP250)定量检测铁调素蛋白。配制检测试剂包括：Calibrator Diluent RD5-26 80mL，Wash Buffer 500mL，Substrate Solution 30mL(显色剂A 15mL与B 15mL等体积混匀后于15分钟内避光使用，每孔的终体积为200μL)，人Hepcidin标准品：重 组人Hepcidin标准品终浓度为1000、500、250、125、62.5、31.3、15.6、0(pg/mL)。每孔加入50μL Assay Diluent RD1-21。每孔加入50μL标准品、对照或样品，盖上密封条，室温孵育2小时。吸出孔中液体，并加入400μL Wash Buffer洗板，共洗4次，每次都要将残液吸净再进行下一次清洗。每孔加入200μL人Hepcidin结合液，盖上新的密封条，室温孵育2小时。吸出孔中液体，并加入400μL Wash Buffer洗板，共洗4次，每次都要将残液吸净再进行下一次清洗。每孔加入200μL Substrate Solution，避光，室温孵育30分钟。每孔加入50μL Stop Solution，孔中颜色应由蓝转黄。如果孔中颜色为绿色或者颜色变化不一致，轻轻敲击反应板以充分混匀。经洗涤、显色后使用酶标仪在450nm测定吸光度值，并根据人铁调素蛋白测定准曲线计算检测样品中铁调素蛋白浓度，用Origin 7.5软件拟合曲线，计算受试化合物抑制HepG2细胞生成铁调素蛋白的的IC50值(nM)。
表2.化合物对HepG2细胞中Hepcidin的抑制活性测试结果。
Figure PCTCN2019128246-appb-000021
*化合物I为依照WO2014151871 A2制备的专利中化合物12。
表1中，A、B、C表示的IC 50值范围分别为：A≤200nM，200nM<B<500nM，C≥500nM。
测试结果表明本发明实施例化合物对HepG2细胞中Hepcidin的表达具有显著的抑制作用。

Claims (16)

  1. 一种式(I)化合物：
    Figure PCTCN2019128246-appb-100001
    其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
    X为-(CR 7R 8) p-；
    R 1选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基，其中烷基、烯基、炔基、杂烷基、环烷基、杂环基、芳基或杂芳基可被任意取代；
    R 2选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9或-NR 9COR 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 3、R 4和R 5分别独立地选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9、-SO 2R 9、-NR 9COR 10、-NR 9CONR 10R 11、-NR 9CO 2R 10、-NR 9SO 2NR 10R 11或-NR 9SO 2R 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 6选自-H、卤素、-OH、-CN、-CF 3、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基或杂烷基，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基、杂烷基、-C 2-6烯基、-C 2-6炔基、饱和或不饱和的杂环基、氧代或3-8元环烷基；其中烷基、烯基、炔基、杂环基或环烷基可被任意取代；
    R 9、R 10和R 11分别独立地选自-H、杂烷基、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基；其中(R 9和R 10)和/或(R 10和R 11)连同与它们相连的氮原子一起形成可被至少一个R 12取代的饱和或不饱和的杂环；
    每个R 12分别独立地选自-H、卤素、-C 1-6烷基或氧代；
    m为0，1，2或3；
    p为1，2或3；
    q为0，1，2或3。
  2. 如权利要求1所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中所述化合物如式(II)所示：
    Figure PCTCN2019128246-appb-100002
    X为-(CR 7R 8) p-；
    R 1选自可被任意取代的芳基或杂芳基；
    R 2选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9或-NR 9COR 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 3、R 4和R 5分别独立地选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9、-SO 2R 9、-NR 9COR 10、-NR 9CONR 10R 11、-NR 9CO 2R 10、-NR 9SO 2NR 10R 11或-NR 9SO 2R 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 6选自-H、卤素、-OH、-CN、-CF 3、-NO 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基或杂烷基，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基、杂烷基、-C 2-6烯基、-C 2-6炔基、饱和或不饱和的杂环基、氧代或3-8元环烷基；其中烷基、烯基、炔基、杂环基或环烷基可被任意取代；
    R 9、R 10和R 11分别独立地选自-H、杂烷基、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基；其中(R 9和R 10)和/或(R 10和R 11)连同与它们相连的氮原子一起形成可被至少一个R 12取代的饱和或不饱和的杂环；
    每个R 12分别独立地选自-H、卤素、-C 1-6烷基或氧代；
    p为1，2或3；
    q为0，1，2或3。
  3. 如权利要求2所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中所述化 合物如式(III)所示：
    Figure PCTCN2019128246-appb-100003
    X为-(CR 7R 8) p-；
    R 1选自可被任意取代的杂芳基；
    R 2选自-H、卤素或-C 1-6烷基；其中烷基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
    R 3和R 4分别独立地选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-(CH 2) qNR 9R 10-、-CONR 9R 10、-NO 2、C 1-6烷基、C 2-6烯基、C 2-6炔基、杂烷基、-OR 9、-COR 9、-COOR 9、-SO 2R 9、-NR 9COR 10、-NR 9CONR 10R 11、-NR 9CO 2R 10、-NR 9SO 2NR 10R 11或-NR 9SO 2R 10，其中烷基、烯基、炔基或杂烷基可被任意取代；
    R 6选自-H、卤素、-OH、-CN、-CF 3、-NO 2、-C 1-6烷基或杂烷基，其中烷基或杂烷基可被任意取代；
    R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基、杂烷基、-C 2-6烯基、-C 2-6炔基或氧代；其中烷基、烯基或炔基可被选自-H、卤素、-C 1-4烷基的取代基取代；
    R 9、R 10和R 11分别独立地选自-H、杂烷基、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基、3-8元环烷基、饱和或不饱和的杂环基、芳基或杂芳基；其中(R 9和R 10)和/或(R 10和R 11)连同与它们相连的氮原子一起形成可被至少一个R 12取代的饱和或不饱和的杂环；
    每个R 12分别独立地选自-H、卤素、-C 1-6烷基或氧代；
    p为1，2或3；
    q为0，1，2或3。
  4. 如权利要求3所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
    X为-(CR 7R 8) p-；
    R 1选自可被任意取代的吡啶基；
    R 2选自-H、卤素或-C 1-6烷基；其中烷基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
    R 3和R 4分别独立地选自-H、卤素、-C 1-6烷基或杂烷基；其中烷基或杂烷基可被选自-H、卤素、-C 1-4烷基或氧代的取代基取代；
    R 6选自-H、卤素、-OH、-CN、-C 1-6烷基或-C 1-6烷氧基；其中烷基或烷氧基可被选自-H、卤素、-C 1-4 烷基或氧代的取代基取代；
    R 7和R 8分别独立地选自-H、卤素、-C 1-6烷基或-C 1-6烷氧基，其中烷基或烷氧基可被选自-H、卤素或-C 1-4烷基的取代基取代；
    p为1或2。
  5. 如权利要求4所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
    X为-(CR 7R 8) p-；
    R 1为
    Figure PCTCN2019128246-appb-100004
    其中R 13选自-H、卤素、-OH、-CN、-CF 3、-CHF 2、-CH 2F、-OCF 3、-NO 2、-NH 2、-C 1-6烷基、-C 2-6烯基、-C 2-6炔基或杂烷基；
    R 2选自-H、卤素或C 1-6烷基；
    R 3和R 4分别独立地选自-H、卤素、C 1-6烷基或-C 1-6烷氧基，其中烷基或烷氧基可被选自-H、卤素或-C 1-4烷基或氧代的取代基取代；
    R 6选自-H、卤素、-OH、-CN、-C 1-6烷基或-C 1-6烷氧基；
    R 7和R 8分别独立地选自-H、卤素或-C 1-6烷基，其中烷基可被选自-H、卤素或-C 1-4烷基的取代基取代；
    p为1或2。
  6. 如权利要求5所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其中：
    X为-(CR 7R 8) p-；
    R 1为
    Figure PCTCN2019128246-appb-100005
    其中R 13选自-H、卤素或-C 1-4烷基；
    R 2选自-H或-C 1-4烷基；
    R 3选自-H、-C 1-4烷基或-C 1-4烷氧基；
    R 4选自-H或-C 1-4烷基；
    R 6选自-H、卤素、-CN、-C 1-4烷基或-C 1-4烷氧基；
    R 7和R 8分别独立地选自-H或-C 1-4烷基；
    p为1或2。
  7. 如权利要求1至6所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其选自：
    Figure PCTCN2019128246-appb-100006
    Figure PCTCN2019128246-appb-100007
  8. 如权利要求1至7所述的化合物，其药学上可接受的盐、水合物、溶剂化物或立体异构体，其选自：
    Figure PCTCN2019128246-appb-100008
  9. 一种如权利要求1至8中任一项所述式(I)化合物，其立体异构体，互变异构体或药学上可接受的盐的制备方法，包括如下步骤：
    Figure PCTCN2019128246-appb-100009
    其中，R 1、R 2、R 3、R 4、R 5和R 6的定义如权利要求1所述；
    中间体A和中间体B在酸性或者碱性条件下，在合适的溶剂中，在合适的温度下，反应生成式(I)化合物。
  10. 一种药物组合物，其含有治疗有效量的根据权利要求1-8中任意一项所述的化合物，或其药学上可接受的载体、稀释剂或赋形剂。
  11. 根据权利要求1-8中任意一项所述的化合物或其可药用的盐或根据权利要求10所述的药物组合物在制备用于预防和/或治疗具有ALK2激酶介导的病理学特征的疾病的药物中的用途。
  12. 根据权利要求11中的用途，其中所述具有ALK2激酶介导的病理学特征的疾病为贫血。
  13. 根据权利要求11中的用途，其中所述具有ALK2激酶介导的病理学特征的疾病为癌症。
  14. 一种治疗预防和/或治疗预防具有ALK2激酶介导的病理学特征的疾病的方法，其包括向患者施用治疗有效量的权利要求1-8中任意一项所述的化合物或其可药用盐或根据权利要求10所述的药物组合物，其中所述具有ALK2激酶介导的病理学特征的疾病选自贫血或癌症。
  15. 一种治疗预防和/或治疗预防具有ALK2激酶介导的病理学特征的疾病的方法，其包括向患者施用治疗有效量的权利要求1-8中任意一项所述的化合物或其可药用盐或根据权利要求10所述的药物组合物，其中所述具有ALK2激酶介导的病理学特征的疾病为贫血，其中所述的贫血选自慢性病性贫血、慢性炎症贫血、癌症或进行性纤维发育不良贫血。
  16. 一种治疗预防和/或治疗预防具有ALK2激酶介导的病理学特征的疾病的方法，其包括向患者施用治疗有效量的权利要求1-8中任意一项所述的化合物或其可药用盐或根据权利要求10所述的药物组合物，其中所述具有ALK2激酶介导的病理学特征的疾病为癌症，其中所述的癌症选自***癌、乳腺癌、***、子宫内膜癌、结肠癌、胃癌、肺癌、肝癌、膀胱癌、胰腺癌、直肠癌、皮肤癌(包括黑色素瘤和基底细胞癌)、口腔癌、骨癌、卵巢癌、脑癌、头颈部癌、间皮内膜癌、白血病、淋巴瘤、食管癌、肾癌、甲状腺癌、骨髓瘤、绒毛膜癌、睾丸癌、神经胶质瘤、母神经胶质瘤、输卵管肿瘤、骨髓纤维化、真性红细胞增多症或特发性血小板增多症。
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