Pyrimidopyrroles compoundsThe invention claims priority from the prior application entitled "pyrimidopyrroles" filed 30/9/2020 to the national intellectual property agency under patent application number 202011061652.8. The entire contents of the above-mentioned application are incorporated by reference into the present invention.
Technical Field
The present invention relates to pyrimidopyrroles or pharmaceutically acceptable salts thereof, pharmaceutical compositions containing them and their use in the prevention or treatment of kinase related diseases such as Janus kinase (JAK, particularly JAK 3) and/or Bruton's tyrosine kinase, BTK related diseases.
Background
Autoimmune diseases are a group of diseases that cause attack on cells or tissues of the body by immune dysfunction, resulting in inflammation and tissue damage, including rheumatoid arthritis (Rheumatoid arthritis, RA), inflammatory bowel disease (Inflammatory bowel disease, IBD), and systemic lupus erythematosus (Systemic lupus erythematosus, SLE), among others. BTK and JAK3 are two important targets for autoimmune diseases.
BTK is a member of the TEC family of non-receptor tyrosine kinases structurally comprising a PH domain, a TH domain, an SH3 domain, an SH2 domain and an SH1 domain. BTK plays a key role in the activation process of B cell antigen receptor (B CELL ANTIGEN receptor, BCR) signaling pathway, regulates the development and activation of B cells, plays an important role in proliferation of B cells, expression of pro-inflammatory cytokines and secretion of antibodies (TARGETING BRUTON's tyrosine kinase in B cell macronancies. Nat Rev cancer.2014Apr;14 (4): 219-32), and therefore BTK becomes one of important targets for treating diseases related to abnormal activation of B cells, including autoimmune diseases and B cell lymphomas. Ibrutinib, acalabrutinib and Zanubrutinib are three BTK inhibitors that have been batched, mainly to treat B-cell lymphomas, with significant efficacy in some patients, but serious side effects and drug-resistant mutations are also clinically observed. Ibrutinib in 2017 was approved by the us FDA for the treatment of graft versus host disease (Graft versus host disease, GVHD), while other BTK inhibitors are currently being actively explored clinically for the treatment of autoimmune diseases, including RA, SLE and multiple sclerosis (Multiple sclerosis, MS).
JAK3 is a member of the JAK family of non-receptor tyrosine kinases. The JAK kinase family has 4 members, JAK-1, JAK-2, JAK-3 and TYK-2.STAT is a downstream substrate of JAK3, and JAK3 activates STAT to dimer into the nucleus, regulating transcriptional expression of specific genes. JAK-STAT signaling pathway plays an important role (JAK inhibition as a therapeutic strategy for immune and inflammatory diseases.Nat Rev Drug Discov.2017December 28;17(1):78;The JAK-STAT Pathway:Impact on Human Disease and Therapeutic Intervention.Annual Review of Medicine.Vol.66:311-328), in lymphocyte proliferation, differentiation, and expression of pro-inflammatory cytokines and JAK3 is one of the targets for autoimmune diseases and malignant tumors. Tofacitinib is an FDA approved JAK3 inhibitor that exhibits good clinical efficacy in RA and IBD. However, there are also adverse effects, including severe infection, liver injury, etc., which are believed to be associated with inadequate selectivity of Tofacitinib for JAK1/2 (JAK inhibition as a therapeutic strategy for immune and inflammatory diseases.Nat Rev Drug Discov.2017December 28;17(1):78;JAK-inhibitors.New players in the field of immune-mediated diseases,beyond rheumatoid arthritis.Rheumatology(Oxford).2019Feb1;58(Suppl 1):i43-i54).
In addition to the individual clinical effects of BTK and JAK3 inhibitors, inhibition of the BTK/JAK3 signaling pathway would exhibit synergistic efficacy. Several studies showed that simultaneous inhibition of BTK and JAK in collagen-induced rat arthritis model (CIA) showed significant relief of joint swelling, reduced osteoclast number, significantly improved pathology scores, and better efficacy than single drug action (2016 ACR/ARHP Annual meeting. Abstraction 484;2013ACR/ARHP Annual meeting. Abstraction 2353). Abbvie initiated clinical secondary experiments on RA and SLE at ABBV599 (BTK inhibitor and JAK inhibitor combination) at 2018, 9 and 2019, 6, respectively. Another dual target inhibitor DWP212525 against BTK/JAK3 also showed disease remission and joint protection in the mouse CIA model (2019 ACR/ARHP Annual meeting. Abstracting 965).
In view of the huge autoimmune disease market and unmet market demand, based on the functions of BTK and JAK3 on autoimmune diseases and the existing clinical effects, it is necessary to develop a double-target small molecule inhibitor with good activity against BTK and JAK3, good selectivity and low toxic and side effects.
Disclosure of Invention
The invention provides a compound shown in a formula (I) or pharmaceutically acceptable salt thereof:
Wherein:
Ring Q is a 5 membered heteroaryl group containing at least 1 atom other than C, N as a ring atom;
X is selected from NH or O;
r1 is selected from H, =o, or the following groups optionally substituted with Ra: c1-C10 alkyl, C3-C14 cycloalkyl, 3-14 membered heterocyclyl, C6-C10 aryl or 5-10 membered heteroaryl;
Ra is selected from F, cl, br, I, OH, CN, = O, NO2 or the following optionally substituted with Rb: NH2、SH、S(O)NH2、S(O)(C1-C10 alkyl), S (O)2(C1-C10 alkyl), P (O) (C1-C10 alkyl), C1-C10 alkyl, C3-C14 cycloalkyl, 3-14 membered heterocyclyl, C1-C10 alkoxy, C3-C14 cycloalkyloxy, 3-14 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy, or 5-10 membered heteroaryloxy;
Rb is selected from F, cl, br, I, OH, CN, = O, NO2、NH2、SH、C1-C10 alkyl, C3-C14 cycloalkyl, 3-14 membered heterocyclyl, C1-C10 alkoxy, C3-C14 cycloalkyloxy, 3-14 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy, or 5-10 membered heteroaryloxy;
r2 is selected from hydrogen, F, cl, br, I, CN, OH, NO2 or the following optionally substituted with Rc: NH2、SH、C1-C10 alkyl, C3-C14 cycloalkyl, 3-14 membered heterocyclyl, C1-C10 alkoxy, C3-C14 cycloalkyloxy, 3-14 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy, or 5-10 membered heteroaryloxy;
Rc is selected from F, cl, br, I, OH, CN, = O, NO2, or the following optionally substituted with Rd: NH2、SH、C1-C10 alkyl, C3-C14 cycloalkyl, 3-14 membered heterocyclyl, C1-C10 alkoxy, C3-C14 cycloalkyloxy, 3-14 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy, or 5-10 membered heteroaryloxy;
R3 is selected from H, F, cl, br, I or C1-C10 alkyl optionally substituted with a group selected from F, cl, br, I, OH;
R4 is selected from H, F, cl, br, I, OH, CN or the following optionally substituted with Rd: c1-C10 alkyl, C1-C10 alkoxy;
Rd is selected from F, cl, br, I, OH;
n is selected from 0 or 1;
the conditions are as follows: when n is 1, R4 is not F, and R2 is not Cl or H.
In some embodiments, X is selected from NH.
In some embodiments, X is selected from O.
In some embodiments, ring Q is a 5 membered heteroaryl group containing 1N atom and 1 atom other than C, N as ring atoms.
In some embodiments, ring Q is selected from thiazolyl, isothiazolyl, oxazolyl, or isoxazolyl.
In some embodiments of the present invention, in some embodiments,Selected from the group consisting ofWherein is representative ofA site of attachment to NH.
In some embodiments of the present invention, in some embodiments,Selected from the group consisting ofWherein is representative ofA site of attachment to NH.
In some embodiments of the present invention, in some embodiments,Selected from the group consisting ofWherein is representative ofA site of attachment to NH.
In some embodiments of the present invention, in some embodiments,Selected from the group consisting ofWherein is representative ofA site of attachment to NH.
In some embodiments, R2 is selected from hydrogen, F, cl, br, I, CN, OH, NO2, or the following groups optionally substituted with Rc: NH2、SH、C1-C6 alkyl, C3-C10 cycloalkyl.
In some embodiments, R2 is selected from hydrogen, F, cl, br, I, CN, or the following groups optionally substituted with Rc: c1-C6 alkyl, C3-C10 cycloalkyl.
In some embodiments, Rc is selected from F, cl, br, I, OH, CN, = O, NO2, or the following groups optionally substituted with Rd: NH2、SH、C1-C10 alkyl, C3-C10 cycloalkyl, 3-10 membered heterocyclyl, C1-C10 alkoxy, C3-C10 cycloalkyloxy, 3-10 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy or 5-10 membered heteroaryloxy.
In some embodiments, R2 is selected from F, cl, br, I or C3-C6 cycloalkyl.
In some embodiments, R2 is selected from F, cl or cyclopropyl.
In some embodiments, R2 is selected from F, cl, br, or I.
In some embodiments, R2 is selected from F or Cl.
In some embodiments, R2 is selected from F or cyclopropyl.
In some embodiments, R2 is selected from cyclopropyl.
In some embodiments, R3 is selected from H, F, cl, br, I or C1-C6 alkyl optionally substituted with a group selected from F, cl, br, I, OH.
In some embodiments, R3 is selected from H, F, cl, br, I or C1-C4 alkyl optionally substituted with a group selected from F, cl, br, I, OH.
In some embodiments, R3 is selected from H, F, cl, br or I.
In some embodiments, R3 is selected from H.
In some embodiments, R1 is selected from H or the following groups optionally substituted with Ra: c1-C10 alkyl, C3-C10 cycloalkyl, 3-10 membered heterocyclyl, C6-C10 aryl or 5-10 membered heteroaryl.
In some embodiments, R1 is selected from C1-C10 alkyl or 3-10 membered heterocyclyl, optionally substituted with Ra.
In some embodiments, R1 is selected from C1-C6 alkyl or 4-6 membered heterocyclyl, optionally substituted with Ra.
In some embodiments, R1 is selected from C1-C6 alkyl or 4-6 membered heterocyclyl containing O and/or N as ring atoms, said C1-C6 alkyl or 4-6 membered heterocyclyl optionally being substituted with Ra.
In some embodiments, Ra is selected from F, cl, br, I, OH, CN, = O, NO2, or the following groups optionally substituted with Rb: NH2、SH、C1-C10 alkyl, C3-C10 cycloalkyl, 3-10 membered heterocyclyl, C1-C10 alkoxy, C3-C10 cycloalkyloxy, 3-10 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy or 5-10 membered heteroaryloxy.
In some embodiments, Rb is selected from F, cl, br, I, OH, CN, = O, NO2、NH2、SH、C1-C10 alkyl, C3-C10 cycloalkyl, 3-10 membered heterocyclyl, C1-C10 alkoxy, C3-C10 cycloalkyloxy, 3-10 membered heterocyclyloxy, C2-C10 alkenyl, C2-C10 alkynyl, C6-C10 aryl, 5-10 membered heteroaryl, C6-C10 aryloxy, or 5-10 membered heteroaryloxy.
In some embodiments, R1 is selected from C1-C6 alkyl.
In some embodiments, R1 is selected from methyl.
In some embodiments, R4 is selected from H, F, cl, br, I, OH, CN or the following groups optionally substituted with Rd: c1-C10 alkyl.
In some embodiments, R4 is selected from H, F, cl, br, I, OH, CN or C1-C6 alkyl.
In some embodiments, R4 is selected from H or C1-C6 alkyl.
In some embodiments, R4 is selected from H or methyl.
In some embodiments, n is selected from 1.
In some embodiments, n is selected from 0.
In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the group consisting of a compound of formula (II):
Wherein X, R1、R2、R3、R4 and n are as defined above.
In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the group consisting of a compound of formula (III):
Wherein X, R1、R2、R3、R4 and n are as defined above.
In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (Ia):
Wherein X, Q, R1、R2、R3、R4 and n are as defined above.
In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIa):
wherein X, R1、R2、R3、R4 and n are as defined above.
In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIIa):
wherein X, R1、R2、R3、R4 and n are as defined above.
In some embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is selected from the following compounds, or pharmaceutically acceptable salts thereof:
The invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, a pharmaceutically acceptable carrier and/or excipient thereof.
Further, the present invention relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament for preventing or treating a disease associated with Janus kinase (JAK, particularly JAK 3) and/or Bruton's Tyrosine Kinase (BTK).
Further, the present invention relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing or treating a Janus kinase (JAK, particularly JAK 3) and/or Bruton's Tyrosine Kinase (BTK) related disease.
Further, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing or treating a disease associated with Janus kinase (JAK, particularly JAK 3) and/or Bruton's Tyrosine Kinase (BTK).
The present invention also relates to a method of treating a disease associated with Janus kinase (JAK, particularly JAK 3) and/or Bruton's Tyrosine Kinase (BTK), which method comprises administering to a patient a therapeutically effective dose of a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein.
Preferred embodiments of the present invention wherein the Janus kinase (JAK, particularly JAK 3) and/or Bruton Tyrosine Kinase (BTK) related diseases include, but are not limited to, neoplasms, autoimmune diseases, and the like.
Definition and description of terms
Unless otherwise indicated, the radical and term definitions recited in the specification and claims of the present invention, including as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and combinations of radical definitions and structures of compounds should fall within the scope of the present description. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.
The term "stereoisomer" refers to an isomer produced by the spatial arrangement of atoms in a molecule, and includes cis-trans isomers, enantiomers, non-corresponding isomers and conformational isomers.
The compounds of the invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, enantiomers, diastereomers, geometric isomers and individual isomers are all included within the scope of the present invention. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms may be present in the substituents such as alkyl groups, and all such isomers and mixtures thereof are included within the definition of compounds of the invention. The asymmetric atom-containing compounds of the present invention may be isolated in optically pure form or in racemic form, which may be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.
The graphic representation of racemates or enantiomerically pure compounds herein is from Maehr, J.chem. Ed.1985, 62:114-120. Unless otherwise indicated, wedge-shaped keys and dotted keys are usedAnd) Representing the absolute configuration of a stereogenic center. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, they include E, Z geometric isomers unless specified otherwise. Likewise, all tautomeric forms are included within the scope of the invention.
The compounds of the invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present invention.
The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The location of the equilibrium depends on the chemical nature of the molecule. For example, among many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type predominates; whereas, among phenols, the enol form is dominant. The present invention encompasses all tautomeric forms of the compounds.
The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =o), meaning that two hydrogen atoms are substituted, oxo does not occur on the aromatic group.
The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl "optionally" substituted with halogen means that ethyl can be unsubstituted (CH2CH3), monosubstituted (e.g., CH2CH2 F), polysubstituted (e.g., CHFCH2F、CH2CHF2, etc.), or fully substituted (CF2CF3). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, no substitution or pattern of substitution is introduced that is sterically impossible and/or synthetic.
When any variable (e.g., Ra or Rb) occurs more than once in the composition or structure of a compound, its definition in each case is independent. For example, if one group is substituted with 2Rb, then each Rb has an independent option.
When the number of one linking group is 0, such as- (CH2)0) -indicates that the linking group is a bond.
When one of the variables is selected from the group consisting of a chemical bond or is absent, the two groups representing its attachment are directly linked, e.g., when L in A-L-Z represents a bond, it is meant that the structure is actually A-Z.
The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.
Cm-Cn herein refers to an integer number of carbon atoms in the m-n range.
The term "alkyl" refers to a hydrocarbon group of the formula CnH2n+1, which may be straight or branched. The term "C1-C10 alkyl" is understood to mean a straight or branched saturated monovalent hydrocarbon radical having 1,2, 3,4,5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, etc.; "C1-C6 alkyl" is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1,2, 3,4,5, 6 carbon atoms.
The term "alkoxy" refers to a monovalent group generated by the loss of a hydrogen atom on a hydroxyl group of a straight or branched chain alcohol, and is understood to be "alkyloxy" or "alkyl-O-". The term "C1-C10 alkoxy" is understood to mean "C1-C10 alkyloxy" or "C1-C10 alkyl-O-".
The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, straight or branched chain, and having at least one double bond. The term "C2-C10 alkenyl" is understood to mean a straight-chain or branched unsaturated monovalent hydrocarbon radical which contains one or more double bonds and has 2, 3,4,5, 6, 7, 8, 9 or 10 carbon atoms, "C2-C10 alkenyl" is preferably "C2-C6 alkenyl", more preferably "C2-C4 alkenyl", even more preferably C2 or C3 alkenyl. It will be appreciated that where the alkenyl group comprises more than one double bond, the double bonds may be separated or conjugated to each other. Specific examples of the alkenyl group include, but are not limited to, vinyl, allyl, (E) -2-methylvinyl, (Z) -2-methylvinyl, (E) -but-2-enyl, (Z) -but-2-enyl, (E) -but-1-enyl, (Z) -but-1-enyl, isopropenyl, 2-methylpropan-2-enyl, 1-methylpropan-2-enyl, 2-methylpropan-1-enyl, (E) -1-methylpropan-1-enyl, or (Z) -1-methylpropan-1-enyl, and the like.
The term "alkynyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms having at least one triple bond. The term "C2-C10 alkynyl" is understood to mean a straight or branched unsaturated monovalent hydrocarbon radical containing one or more triple bonds and having 2, 3,4,5, 6,7,8, 9 or 10 carbon atoms. Examples of "C2-C10 alkynyl" include, but are not limited to, ethynyl (-C.ident.CH), propynyl (-C.ident.CCH3、-CH2 C.ident.CH), but-1-ynyl, but-2-ynyl, or but-3-ynyl. "C2-C10 alkynyl" may include "C2-C3 alkynyl" and examples of "C2-C3 alkynyl" include ethynyl (-C.ident.CH), prop-1-ynyl (-C.ident.CCH3), prop-2-ynyl (propargyl).
The term "cycloalkyl" refers to a fully saturated carbocycle in the form of a single ring, a parallel ring, a bridged ring, or a spiro ring. The term "C3-C14 cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 14 carbon atoms. The term "C3-C10 cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 10 carbon atoms. The term "C3-C6 cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3,4,5, 6 carbon atoms. Examples of cycloalkyl radicals are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or bicyclic hydrocarbon radicals such as the decalin ring. According to the invention, the bicyclic hydrocarbon ring includes a bridged, spiro or fused ring structure.
The term "cycloalkyloxy" is understood as "cycloalkyl-O-". The term "cycloalkyloxy" is understood as "cycloalkyl-O-".
The term "heterocyclyl" refers to a fully saturated or partially saturated (wholly not aromatic heteroaromatic) monovalent monocyclic, fused, spiro, or bridged ring radical containing 1-5 heteroatoms or groups of heteroatoms (i.e., groups containing heteroatoms) in the ring atoms, including but not limited to nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), S (=o)2 -, -S (=o) -and optionally substituted-NH-, -S (=o) (=nh) -, -C (=o) NH-, -C (=nh) -, -S (=o)2 NH-, S (=o) NH-, or-NHC (=o) NH-, and the like. The term "3-14 membered heterocyclyl" is understood to mean a saturated or partially saturated monovalent mono-or bicyclic hydrocarbon ring having 3 to 14 ring atoms, which contains 1 to 5, preferably 1 to 3 heteroatoms selected from N, O and S. The term "3-10 membered heterocyclyl" means a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring comprising 1 to 5, preferably 1 to 3 heteroatoms selected from N, O and S. The term "4-6 membered heterocyclyl" is understood to mean a saturated or partially saturated monovalent mono-or bicyclic hydrocarbon ring having 4,5, 6 ring atoms, which contains 1 to 5, preferably 1 to 3 heteroatoms selected from N, O and S. In particular, the heterocyclic groups may include, but are not limited to: 4-membered rings such as azetidinyl, oxetanyl; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a6 membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a partially saturated 6-membered ring such as tetrahydropyridinyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclyl may be benzo-fused. The heterocyclyl may be bicyclic, such as, but not limited to, a5, 5 membered ring, such as hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl ring, or a5, 6 membered bicyclic ring, such as hexahydropyrrolo [1,2-a ] pyrazin-2 (1H) -yl ring. The ring containing nitrogen atoms may be partially unsaturated, i.e. it may contain one or more double bonds, such as but not limited to 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4] thiadiazinyl, 4, 5-dihydro-oxazolyl or 4H- [1,4] thiazinyl, or it may be benzo-fused, such as but not limited to dihydroisoquinolinyl. According to the invention, the heterocyclic group is non-aromatic. The bicyclic hydrocarbon ring includes a bridged, spiro, or fused ring structure.
The term "heterocyclyloxy" is understood to mean "heterocyclyl-O-".
The term "aryl" refers to an all-carbon monocyclic or fused-polycyclic aromatic ring radical having a conjugated pi-electron system. The term "C6-C20 aryl" is understood to mean preferably a mono-, bi-or tricyclic hydrocarbon ring of monovalent aromatic or partly aromatic nature having from 6 to 20 carbon atoms. In particular a ring having 6 carbon atoms ("C6 aryl"), such as phenyl; or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms ("C13 aryl"), such as fluorenyl, or a ring having 14 carbon atoms ("C14 aryl"), such as anthracenyl. "C6-C10 aryl" is understood to mean a monovalent aromatic or partially aromatic, monocyclic, bicyclic or tricyclic hydrocarbon ring having 6, 7, 8, 9, 10 carbon atoms, in particular a ring having 6 carbon atoms ("C6 aryl"), for example phenyl; or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl.
The term "aryloxy" may be understood as "aryl-O-".
The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic ring system containing at least one ring atom selected from N, O, S and the remaining ring atoms being aromatic ring groups of C. The term "5-20 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: having 5 to 20 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S, for example "5-14 membered heteroaryl". The term "5-14 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: it has 5, 6,7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and it contains 1 to 5, preferably 1 to 3, heteroatoms which are each independently selected from N, O and S and which may additionally be benzo-fused in each case. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl and the like, and their benzo derivatives, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazole, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, and the like; or an axcinyl group, an indolizinyl group, a purinyl group, etc., and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like. "5-10 membered heteroaryl" is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring system having 5, 6,7, 8, 9, 10 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S.
The term "heteroaryloxy" may be understood as "heteroaryl-O-".
The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, organic acids and bases.
The term "pharmaceutical composition" means a mixture of one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
The term "therapeutically effective amount" means an amount of a compound of the invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and disclosure.
The term "adjuvant" refers to a pharmaceutically acceptable inert ingredient. Examples of the category of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical formulation, i.e., by increasing flowability and/or tackiness, making the formulation more suitable for direct compression. Typical examples of "pharmaceutically acceptable carriers" suitable for use in the above formulations are: saccharides, starches, cellulose and derivatives thereof, and the like.
The words "comprise", "comprising" or "includes" and variations thereof such as include or comprise are to be interpreted in an open, non-exclusive sense, i.e. "including but not limited to".
The invention also includes isotopically-labeled compounds of the invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I and36 Cl, respectively, and the like.
Certain isotopically-labeled compounds of the present invention (e.g., labeled with3 H and14 C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e.,3 H) and carbon-14 (i.e.,14 C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as15O、13N、11 C and18 F are useful in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below by substituting an isotopically-labeled reagent for an non-isotopically-labeled reagent.
The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.
The chemical reactions of the embodiments of the present invention are accomplished in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the embodiments already present.
Detailed Description
The following examples illustrate the technical aspects of the invention in detail, but the scope of the invention is not limited thereto.
All reagents used in the present invention are commercially available and can be used without further purification. Unless otherwise indicated, the ratio of the mixed solvent is a volume mixing ratio. Unless otherwise indicated,% refers to wt%.
The compounds being obtained by hand or by handSoftware naming, commercial compounds are referred to by vendor catalog names.
The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). The unit of NMR shift was 10-6 (ppm). The solvent for NMR measurement is deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is Tetramethylsilane (TMS); "IC50" refers to half the inhibitory concentration, meaning the concentration at which half the maximum inhibitory effect is achieved; EA: ethyl acetate; PE: petroleum ether; THF: tetrahydrofuran; DMF: n, N-dimethylformamide; TFA: trifluoroacetic acid; SEM: me3SiCH2CH2OCH2 -; boc: t-butoxycarbonyl; DIPEA: n, N-diisopropylethylamine; DMSO: dimethyl sulfoxide; pd2(dba)3: tris (dibenzylideneacetone) dipalladium; pd (dppf) Cl2: [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride; BINAP:1,1 '-binaphthyl-2, 2' -bisdiphenylphosphine; selectFluor: N-fluoro-N' - (chloromethyl) triethylenediamine bis (tetrafluoroborate); TLC: thin layer chromatography; NIS: n-iodosuccinimide; DCM: dichloromethane.
EXAMPLE 1 preparation of Compound 001
1- ((2 S,4 r) -4- ((5-chloro-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one
Synthetic route and specific synthetic steps:
the first step: synthesis of 2,4, 5-trichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 1-b
2,4, 5-Trichloro-7H-pyrrolo [2,3-d ] pyrimidine (16.7 g,75.6 mmol) was dissolved in dry tetrahydrofuran (170.0 mL), sodium hydride (3.9 g,90.7 mmol) was added under nitrogen at zero degrees Celsius, and after stirring at zero degrees Celsius for 0.5H, 2- (trimethylsilyl) ethoxymethyl chloride (15.1 g,90.7 mmol) was slowly added dropwise, followed by stirring at room temperature for 12H. Saturated brine was added to quench, the product was extracted with EA, and after concentration of the organic phase, the crude product was passed through a silica gel column (ethyl acetate: petroleum ether=100:1 to 30:1) to give 1-b (19.0 g, yield: 71.7%).
LCMS:Rt:2.180min；MS m/z(ESI):352.1[M+H].
And a second step of: synthesis of tert-butyl (2S, 4R) -4- ((2, 5-dichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylate 1-d
2,4, 5-Trichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 1-b (526 mg,1.50 mmol), (2S, 4R) -4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester 1-c (300 mg,1.5 mmol) and DIPEA (284 mg,3.75 mmol) were added to a solution of isopropanol (15.0 mL) and stirred at 90℃for 16 hours. After completion of the reaction by TLC, the organic phase was concentrated under reduced pressure and passed through a silica gel column (ethyl acetate: petroleum ether=5:1) to give 1-d (700 mg, yield: 92.3%).
LCMS:Rt:2.357min；MS m/z(ESI):516.1[M+H].
And a third step of: synthesis of tert-butyl (2S, 4R) -4- ((5-chloro-2- ((3-methylisothiazol-5-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylate 1-f
Pd2(dba)3 (36.0 mg,0.038 mmol) was added to a solution of 1-d (200 mg, 0.3838 mmol), 3-methylisothiazol-5-amine 1-e (75 mg,0.50 mmol), BINAP (24 mg,0.038 mmol) and cesium carbonate (316 mg,0.97 mmol) in 1, 4-dioxane (20.0 mL) at room temperature under nitrogen, and the reaction was warmed to 120℃and stirred overnight. Cooled to room temperature, filtered, and the reaction solution was concentrated under reduced pressure and purified by silica gel column (petroleum ether: ethyl acetate=2:1) to give 1-f (193 mg, yield: 83.6%)
LCMS:Rt:1.795min；MS m/z(ESI):594.6[M+H].
1H NMR(400M Hz,DMSO-d6)δ11.03(s,1H),7.40(s,1H),6.71(s,1H),6.60-6.53(m,1H),555(s,2H),4.79(brs,1H),3.99-3.93(m,2H),3.64(s,2H),3.34(s,1H),2.36(s,3H),2.22(s,1H),1.92(s,1H),1.51(s,9H),1.40(d,J＝6.4Hz,3H),0.93(t,J＝8.0Hz,2H),0.00(s,9H).
Fourth step: synthesis of 5-chloro-N2 - (3-methylisothiazol-5-yl) -N4 - ((3R, 5S) -5-methylpyrrolidin-3-yl) -7H-pyrrolo [2,3-d ] pyrimidine-2, 4-diamine 1-g
1-F (193 mg,0.33 mmol) was added to a dichloromethane solution (5.0 mL) of trifluoroacetic acid (0.5 mL) at room temperature, and the reaction was stirred at 0deg.C for 16 hours. The reaction was concentrated to dryness under reduced pressure, and the crude product was added to LiOH (133 mg,10.0 mmol) in THF/H2 O (4.0/1.0 mL) without purification, and the reaction was stirred at 25℃for 2 hours. The reaction solution was concentrated under reduced pressure and subjected to FLASH (acetonitrile/water/1%TFA) to give 1-g (71 mg, yield: 59%).
LCMS:Rt:1.15min；MS m/z(ESI):364.2[M+H]。
Fifth step: synthesis of 1- ((2S, 4R) -4- ((5-chloro-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one 001
1-G (70 mg,0.19 mmol) was dissolved in a mixture of tetrahydrofuran (5.0 mL) and water (2.0 mL) at 0deg.C, and after adding a potassium phosphate solid (100 mg,0.37 mmol), a solution of acryloyl chloride (17 mg,0.19 mmol) in tetrahydrofuran (1.0 mL) was added dropwise and stirred at 25deg.C for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by Prep-HPLC (acetonitrile/water/1%HCO2 H) to give 001 (21.1 mg, yield: 12.5).
LCMS:Rt:7.154min；MS m/z(ESI):418.1[M+H].
1H NMR(400M Hz,DMSO-d6)δ11.72(s,1H),11.01(s,1H),7.12(d,J＝2.4Hz,1H),6.59-6.51(m,3H),6.15(t,J＝16.8Hz,1H),5.69-5.63(m,1H),4.77(s,1H),4.28(s,1H),3.94(s,2H),3.60-3.40(m,1H),2.28(s,3H),1.99-1.86(m,1H),1.34(d,J＝6.0Hz,3H).
EXAMPLE 2 preparation of Compound 002
1- ((2 S,4 r) -4- ((5-chloro-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -2-methylpyrrolidin-1-yl) prop-2-en-1-one
Synthetic route and specific synthetic steps:
the first step: synthesis of tert-butyl (2S, 4R) -4- ((2, 5-dichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -2-methylpyrrolidine-1-carboxylate 2-c
NaH (51.2 mg,1.28 mmol) was added in portions to a solution of (2S, 4R) -tert-butyl 4-hydroxy-2-methylpyrrolidine-1-carboxylate 2-b (300 mg,1.16 mmol) in DMSO (4.0 mL) at room temperature, stirring was completed for 15 min, then 2,4, 5-trichloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H pyrrolo [2,3-d ] pyrimidine 2-a (408 mg,1.16 mmol) was added, the reaction system warmed to 55℃for 15 min, 50.0mL of saturated NH4 Cl solution was added to the reaction solution, three times (50.0 mL of 3) were extracted with dichloromethane, the organic phases were combined, each time washed with water (50.0 mL) and saturated brine (50.0 mL), the organic phase was dried over anhydrous sodium sulfate for 1 hour, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (ethyl acetate: petroleum ether=1:8) to give pure product 2-c (532 mg: yield: 532%).
LCMS:Rt:8.337min；MS m/z(ESI):517.1，519.1[M+H]。
1H NMR(400MHz,CDCl3)δ7.14(s,1H),5.54(s,2H),4.18-4.09(m,1H),3.86(s,1H),3.78-3.65(m,1H),3.55(t,J＝8.4Hz,2H),2.47(s,1H),2.09-2.06(m,1H),1.50(s,9H),1.45-1.44(m,3H),1.36-1.28(m,1H),0.95(t,J＝8.0Hz,2H),0.00(s,9H).
And a second step of: synthesis of tert-butyl (2S, 4R) -4- ((5-chloro-2- ((3-methylisothiazol-5-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -2-methylpyrrolidine-1-carboxylate 2-e
Pd2(dba)3 (97 mg,0.106 mmol) and BINAP (61 mg,0.106 mmol) were added to a solution of 2-c (543.7 mg,1.05 mmol), 3-methylisothiazol-5-amine hydrochloride (2-d, 240mg,1.59 mmol) and Cs2CO3 (691 mg,2.12 mmol) in 1, 4-dioxane (15.0 mL) under nitrogen at room temperature, and the reaction was warmed to 100℃and stirred for 16 hours, cooling to room temperature. The mixture was filtered, and the cake was washed with 50.0mL of tetrahydrofuran, and the crude product obtained after concentration of the solution under reduced pressure was purified by Chem-flash to give pure 2-e (493 mg, yield: 78.7%).
LCMS:Rt:7.447min；MS m/z(ESI):595.3，597.3[M+H]。
And a third step of: synthesis of N- (5-chloro-4- (((3R, 5S) -5-methylpyrrolidin-3-yl) oxy) -7H-pyrrolo [2,3-d ] pyrimidin-2-yl) -3-methylisothiazol-5-amine 2-f
2-E (243 mg, 0.390 mmol) was added to a dichloromethane solution (3.0 mL) of trifluoroacetic acid (5.0 mL) at room temperature, and the reaction was stirred at room temperature for 1 hour. Concentrated under reduced pressure, and the crude product was added to a tetrahydrofuran/water solution (4.0 mL/1.0 mL) of LiOH.H2 O (50.6 mg, 1.183mmol) without purification, and the reaction was stirred at 25℃for 1 hour. The reaction solution was concentrated under reduced pressure to give crude product, which was purified by CHEMFLASH to give pure 2-f (38 mg, yield: 26.8%).
LCMS:Rt:0.877min；MS m/z(ESI):365.0,367.0[M+H]。
Fourth step: synthesis of 1- ((2S, 4R) -4- ((5-chloro-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) -2-methylpyrrolidin-1-yl) prop-2-en-1-one 002
2-F (38 mg,0.104 mmol) was dissolved in a mixture of tetrahydrofuran (2.0 mL) and water (0.2 mL), and after adding a potassium phosphate solid (22 mg,0.104 mmol), the mixture was stirred at 0℃to which a solution of acryloyl chloride (18.7 mg,0.208 mmol) in tetrahydrofuran (1.0 mL) was added dropwise and the mixture was stirred at 0℃for 1 hour. The reaction solution was concentrated under reduced pressure and purified by Prep-HPLC (acetonitrile/water/1%o HCOOH) to give 002 (15.8 mg, yield: 36.2%, purity: 99.69%).
LCMS:Rt:6.387min；MS m/z(ESI):419.0[M+H]。
1H-NMR(400MHz,DMSO-d6)δ7.30(s,1H),6.70-6.52(m,2H),6.21(dd,J＝4,16Hz,1H),5.88(s,1H),5.76-5.63(dd,J＝4,16Hz,1H),4.44-4.27(dq,J＝8,16Hz,1H),4.02-3.71(m,5H),2.34(s,3H),2.15(dd,J＝12,16Hz,1H),1.43-1.39(dd,J＝4,8Hz,3H).
EXAMPLE 3 preparation of Compound 003
1- ((2 S,4 r) -4- ((5-fluoro-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one
Synthetic route and specific synthetic steps:
the first step: synthesis of 2, 4-dichloro-5-fluoro-7H-pyrrolo [2,3-d ] pyrimidine 3-b
2, 4-Dichloro-7H-pyrrolo [2,3-d ] pyrimidine 3-a (10.0 g,42.4 mmol) and SelectFluor (22.4 g,64.0 mmol) were added to a solution of acetonitrile (200.0 mL) and acetic acid (40.0 mL) and stirred at 80℃for 16 hours. After the completion of the reaction by TLC, the organic phase was concentrated under reduced pressure and passed through a silica gel column (petroleum ether: ethyl acetate=5:1) to give 3-b (6.6 g, yield: 60.5%).
LCMS:Rt:1.11min；MS m/z(ESI):206.0,208.0[M+H].
And a second step of: synthesis of 2, 4-dichloro-5-fluoro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 3-c
3-B (6.6 g,32.20.14 mmol) was added to a solution of tetrahydrofuran (150.0 mL) at room temperature under nitrogen, sodium hydride (1.42 g,35.4 mmol) was added in portions at 0deg.C, stirred for half an hour, and SEM-Cl (8.02 g,48.3 mmol) was added dropwise to the reaction solution, followed by a further reaction for 1h. After TLC monitored completion of the reaction, quenched with 50.0mL of water, extracted with ethyl acetate (100.0 mL x 3) and the organic phase concentrated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate=5:1) to give 3-c (8.0 g, yield: 74.2%).
LCMS:Rt:2.180min；MS m/z(ESI):336.2，338.2[M+H].
And a third step of: synthesis of (2S, 4R) -4- ((2-chloro-5-fluoro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylic acid tert-butyl ester 3-e
3-C (600 mg,1.79 mmol), (2S, 4R) -4-amino-2-methylpyrrolidine-1-carboxylic acid tert-butyl ester 3-d (360 mg,1.79 mmol) and DIPEA (578 mg,4.47 mmol) were added to a solution of isopropanol (20.0 mL) and stirred at 110℃for 16h. After TLC monitored completion of the reaction, the organic phase was concentrated under reduced pressure and passed through a silica gel column (petroleum ether: ethyl acetate=5:1) to give 3-e (720 mg, yield: 80.0%).
LCMS:Rt:2.007min；MS m/z(ESI):500.0[M+H].
1H NMR(400MHz,CDCl3)δ6.80(s,1H),5.49(s,1H),5.37(s,1H),4.79(d,J＝7.2Hz,1H),4.00(s,1H),3.55-3.51(m,2H),3.28-3.23(m,1H),2.64-2.61(m,1H),1.51(s,9H),1.38(d,J＝6.0Hz,2H),1.30(d,J＝7.2Hz,2H),0.96-0.91(m,2H),0.00(s,9H).
Fourth step: synthesis of tert-butyl (2S, 4R) -4- ((5-fluoro-2- ((3-methylisothiazol-5-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylate 3-g
Pd2(dba)3 (110 mg,0.12 mmol), BINAP (75 mg,0.12 mmol) was added to a solution of 3-e (600 mg,1.2 mmol), 3-methylisothiazol-5-amine 3-f (270 mg,1.8 mmol) and cesium carbonate (978 mg,3.0 mmol) in 1, 4-dioxane (20.0 mL) under nitrogen at room temperature, and the reaction system was warmed to 120℃and stirred overnight, cooled to room temperature. The reaction solution was concentrated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate=2:1) to give 3-g (289 mg, yield: 41.7%).
LCMS:Rt:1.697min；MS m/z(ESI):578.1[M+H].
Fifth step: synthesis of 5-fluoro-N2 - (3-methylisothiazol-5-yl) -N4 - ((3R, 5S) -5-methylpyrrolidin-3-yl) -7H-pyrrolo [2,3-d ] pyrimidine-2, 4-diamine 3-H
3-G (205 mg,0.35 mmol) was added to a dichloromethane solution (8.0 mL) of trifluoroacetic acid (4.0 mL) at room temperature, and the reaction was stirred at 0deg.C for 6 hours. The reaction was quenched with saturated sodium bicarbonate (50.0 mL), extracted with ethyl acetate, the organic phase concentrated under reduced pressure, and the crude product was added without purification to a tetrahydrofuran/water solution (10.0 mL/2 mL) of lioh.h2 O (149 mg,3.5 mmol) and the reaction stirred at 25 ℃ for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by Prep-FLASH (acetonitrile/water/1%o CF3CO2 H) to give 3-H (75 mg, yield: 61.7%).
LCMS:Rt:1.17min；MS m/z(ESI):348.2[M+H]。
Sixth step: synthesis of 1- ((2S, 4R) -4- ((5-fluoro-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one 003
3-H (75 mg,0.21 mmol) was dissolved in a mixture of tetrahydrofuran (10.0 mL) and water (2.0 mL), and after adding potassium phosphate solid (111 mg,0.52 mmol), a solution of acryloyl chloride (29 mg,0.32 mmol) in tetrahydrofuran (1.0 mL) was added dropwise thereto with stirring at 0℃for 1 hour. The reaction solution was concentrated under reduced pressure and purified by Prep-HPLC (acetonitrile/water/1%HCO2 H) to give 003 (7.18 mg, yield: 8.5%, purity: 98.7%).
LCMS:Rt:7.017min；MS m/z(ESI):402.0[M+H].
1H NMR(400MHz,DMSO-d6)δ10.72(s,1H),7.08(s,1H),6.82(s,1H),6.69-6.56(m,1H),6.54(s,1H),6.16(t,J＝16.4Hz,1H),5.72-5.63(m,1H),4.78(s,1H),4.26-4.07(m,2H),3.52(t,J＝7.6Hz,1H),2.62(s,1H),2.21(s,3H),2.18(s,1H),2.00-1.83(m,1H),1.31(d,J＝6.0Hz,1H).
Example 4 preparation of Compound 004
1- ((2 S,4 r) -4- ((5-chloro-2- ((2-methylthiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one
Synthetic route and specific synthetic steps:
The first step: synthesis of tert-butyl (2S, 4R) -4- ((5-chloro-2- ((2-methylthiazol-5-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidine-1-carboxylate 4-c
Pd2(dba)3 (95 mg,0.105 mmol) and BINAP (91 mg,0.157 mmol) were added to a solution of 4-a (540 mg,1.05 mmol), 2-methylthiazol-5-amine 4-b (178 mg,1.57 mmol) and Cs2CO3 (683 mg,2.1 mmol) in DMF (9.0 mL) under nitrogen at room temperature, and the mixture was subjected to microwave reaction at 90℃for 30 minutes and cooled to room temperature. Purification by reverse phase (condition: acetonitrile/water (0.01% formic acid)) gave pure 4-c (75.7 mg, yield: 12.2%).
LCMS:Rt:1.81min；MS m/z(ESI):594.2，596.2[M+H]。
And a second step of: synthesis of 5-chloro-N4 - ((3R, 5S) -5-methylpyrrolidin-3-yl) -N2 - (2-methylthiazol-5-yl) -7H-pyrrolo [2,3-d ] pyrimidine-2, 4-diamine 4-d
4-C (75.7 mg,0.128 mmol) was added to a dichloromethane solution (3.0 mL) of trifluoroacetic acid (5.0 mL) at room temperature, and the reaction was stirred at room temperature for 1 hour. Concentrated under reduced pressure, and the crude product was added to a tetrahydrofuran/water solution (4.0 mL/1.0 mL) of LiOH.H2 O (26.8 mg,0.64 mmol) without purification, and the reaction was stirred at 25℃for 1 hour. The reaction solution was concentrated under reduced pressure to give a crude product, which was purified by reverse phase purification (condition: acetonitrile/water (0.01% formic acid)) to give pure 4-d (21 mg, yield: 45.3%).
LCMS:Rt:0.872min；MS m/z(ESI):364.3,366.3[M+H]。
And a third step of: synthesis of 1- ((2S, 4R) -4- ((5-chloro-2- ((2-methylthiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -2-methylpyrrolidin-1-yl) prop-2-en-1-one 004
4-D (21 mg,0.058 mmol) was dissolved in a mixture of tetrahydrofuran (5.0 mL) and water (0.5 mL), and after adding potassium phosphate solid (12.3 mg,0.058 mmol), a solution of acryloyl chloride (3.6 mg,0.041 mmol) in tetrahydrofuran (1.0 mL) was added dropwise and stirred at 0℃for 1 hour. The reaction solution was concentrated under reduced pressure and purified by preparative purification (condition: acetonitrile/water/1%HCOOH) to give 004 (2.11 mg, yield: 8.74%).
LCMS:Rt:5.391min；MS m/z(ESI):418.0[M+H]。
1H-NMR(400MHz,DMSO-d6)δ11.60(s,1H),10.44(d,J＝25.2Hz,1H),7.32(d,J＝11.2Hz,1H),7.08(t,J＝2.4Hz,1H),6.70-6.46(m,2H),6.21-6.13(m,1H),5.73-5.63(m,1H),4.74-4.60(m,1H),4.32-4.26(m,1H),4.18-4.10(m,1H),3.56-3.52(m,1H),3.35-3.30(m,1H),2.57(d,J＝18.8Hz,3H),2.03-1.85(m,1H),1.33(t,J＝6.0Hz,3H).
EXAMPLE 5 preparation of Compound 005
(R) -1- (3- ((5-cyclopropyl-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one
Synthetic route and specific synthetic steps:
the first step: synthesis of 2, 4-dichloro-5-iodo-7H-pyrrolo [2,3-d ] pyrimidine 5-b
2, 4-Dichloro-7H-pyrrolo [2,3-d ] pyrimidine (5-a, 30g,159.5 mmol) and NIS (43.0 g,191.48 mmol) were added to a solution of DCM (150.0 mL) and stirred at room temperature for 16 hours. After completion of the reaction by TLC, filtration, washing with DCM afforded a white solid, washing off excess NIS with saturated Na2SO3 solution, drying the organic phase over anhydrous Na2SO4, filtration, and concentration of the filtrate under reduced pressure afforded 5-b (48.0 g, yield: 86.0%).
LCMS:Rt:0.61min；MS m/z(ESI):313.9[M+H].
And a second step of: synthesis of 2, 4-dichloro-5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 5-c
5-B (25 g,79.9 mmol) was added to anhydrous tetrahydrofuran (150.0 mL) under nitrogen at room temperature, sodium hydride (4.2 g,103.8 mmol) was added in portions at 0deg.C, stirred for half an hour, and SEM-Cl (17.3 g,103.8 mmol) was added dropwise to the reaction solution, followed by a further reaction for 1 hour. After TLC monitored completion of the reaction, the reaction solution was slowly poured into 50.0mL of ice water, extracted with ethyl acetate (100.0 mL of 3), the organic phase was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography on silica gel (petroleum ether: ethyl acetate=25:1) to give 5-c (27.6 g, yield: 78.0%).
LCMS:Rt:1.87min；MS m/z(ESI):444.0，446.0[M+H].
1H NMR(400M Hz,DMSO-d6)δ8.22(s,1H),5.63(s,2H),3.61(t,J＝8.0Hz,2H),0.92(t,J＝8.0Hz,2H),0.00(s,9H).
And a third step of: synthesis of 2, 4-dichloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidine 5-d
Pd (dppf) Cl2 (247.4 mg,0.338 mmol) and Ag2 O (392.1 mg,1.69 mmol) were added to a solution of 5-c (1.5 g,3.38 mmol), cyclopropylboronic acid (436.8 mg,5.0 mmol) and potassium phosphate (2.1 g,10.1 mmol) in 1, 4-dioxane (30 mL) under nitrogen at room temperature, and the reaction was warmed to 120℃and stirred for 16 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate=15:1) to give 5-d (1.09 g, yield: 82.0%).
LCMS:Rt:1.28min；MS m/z(ESI):358.1[M+H]。
Fourth step: synthesis of tert-butyl (R) -3- ((2-chloro-5-cyclopropyl-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate 5-f
5-D (500 mg,1.40 mmol), 5-e (336 mg,1.70 mmol) and DIPEA (361 mg,2.80 mmol) were added to a solution of isopropanol (15.0 mL) and stirred at 90℃for 16 h. After TLC monitored completion of the reaction, the organic phase was concentrated under reduced pressure and passed through a silica gel column (petroleum ether: ethyl acetate=5:1) to give 5-f (500 mg, yield: 68.6%).
LCMS:Rt:2.152min；MS m/z(ESI):522.2[M+H].
Fifth step: synthesis of tert-butyl (R) -3- ((5-cyclopropyl-2- ((3-methylisothiazol-5-yl) amino) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidine-1-carboxylate 5-H
Pd2(dba)3 (91 mg,0.10 mmol), BINAP (62 mg,0.10 mmol) were added to a solution of 5-f (500 mg,1.0 mmol), 3-methylisothiazol-5-amine 5-g (372 mg,2.0 mmol) and cesium carbonate (650 mg,2.0 mmol) in 1, 4-dioxane (30.0 mL) at room temperature under nitrogen, and the reaction system was warmed to 120℃and stirred overnight, cooled to room temperature. The reaction solution was concentrated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate=2:1) to give 5-h (580 mg, yield: > 99%).
LCMS:Rt:1.92min；MS m/z(ESI):600.4[M+H].
Sixth step: synthesis of (R) -5-cyclopropyl-N2 - (3-methylisothiazol-5-yl) -N4 - (piperidin-3-yl) -7H-pyrrolo [2,3-d ] pyrimidine-2, 4-diamine 5-i
5-H (580 mg,1.0 mmol) was added to a dichloromethane solution (6.0 mL) of trifluoroacetic acid (3.0 mL) at room temperature, and the reaction was stirred at 0deg.C for 6 hours. The reaction was quenched with saturated sodium bicarbonate (50.0 mL), extracted with ethyl acetate (10.0 x 3 mL), the organic phase concentrated under reduced pressure, and the crude product was added without purification to a tetrahydrofuran/water solution (10 mL/2 mL) of lioh.h2 O (205 mg,5.0 mmol) and the reaction stirred at 25 ℃ for 2 hours. The reaction was concentrated under reduced pressure to give 5-i (350 mg, yield: > 99%).
LCMS:Rt:0.918min；MS m/z(ESI):369.9[M+H]。
Seventh step: synthesis of (R) -1- (3- ((5-cyclopropyl-2- ((3-methylisothiazol-5-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) piperidin-1-yl) prop-2-en-1-one 005
5-I (350 mg,1.0 mmol) was dissolved in a mixture of tetrahydrofuran (10.0 mL) and water (2.0 mL), and after adding potassium phosphate solid (424 mg,2.0 mmol), a solution of acryloyl chloride (135 mg,1.5 mmol) in tetrahydrofuran (1.0 mL) was added dropwise thereto and stirred at 0℃for 1 hour. The reaction mixture was concentrated under reduced pressure and purified by Prep-HPLC (acetonitrile/water/1%HCO2 H) to give 005 (22.68 mg, yield: 5.5%).
LCMS:Rt:5.227min；MS m/z(ESI):424.1[M+H].
1H NMR(400M Hz,DMSO-d6)δ11.14(brs,2H),6.93-6.69(m,1H),6.67(s,1H),6.60(s,1H),6.21-5.99(m,2H),5.61(dd,J＝8,11.2Hz,1H),4.45(s,1H),3.81(s,2H),3.66-3.50(m,2H),2.32(s,3H),2.01(s,1H),1.90(s,2H),1.72(s,1H),1.59(s,1H),0.83-0.78(dt,J＝8,12Hz,2H),0.53-0.52(dt,J＝8,12Hz,2H).
Biological Activity and related Property test cases
Test example 1: BTK kinase Activity inhibition assay
Experimental principle: after incubation of BTK kinase with the compound, it reacts with the substrate under the action of ATP. ADP produced by the reaction was quantified using the ADP-GLO assay kit from Promega corporation, thereby reflecting the enzyme activity.
Experimental instrument: an Echo650 pipetting system from Labcyte corporation; PERKIN ELMER company Envision microplate reader; eppendorf company 5810 centrifuge.
Experimental materials:
The experimental method comprises the following steps: the test compounds were transferred to 384 well plates using an Echo pipetting system and 2 μl/well BTK was added and incubated for 30 min. Then, 3. Mu.L/well of a mixed solution of substrate Poly (4:1 Glu, tyr) and ATP was added to initiate the enzyme reaction. The final concentrations of the compounds were 3-fold diluted in dimethyl sulfoxide (DMSO) starting from 300nM or 100nM, respectively. The final concentration of enzyme in the reaction was 1.7 ng/well, the final concentration of ATP was 36. Mu.M, and the final concentration of substrate was 0.1mg/mL. After 1 hour of reaction, 5. Mu.L/well ADP-GLO reagent was added and incubated for 40 minutes. Then 10. Mu.L/well kinase reaction detection reagent was added and incubated for 30 minutes. Fluorescence signals were read with an Envision microplate reader and the inhibition ratio, half inhibition concentration (IC50) was calculated.
The biological activity of the compounds of the invention was determined by the above assay and the IC50 values determined are shown in Table 1 below.
TABLE 1 IC for inhibition of BTK kinase Activity by the inventive Compounds50
| Examples compound numbering | IC50(nM) |
| 001 | 1.54 |
| 002 | 14.34 |
| 003 | 1.61 |
| 004 | 4.94 |
| 005 | 9.14 |
Test example 2: JAK3 kinase Activity inhibition assay
Experimental principle: after incubation of JAK3 kinase with the compound, it reacts with the substrate under the action of ATP. ADP produced by the reaction was quantified using the ADP-GLO assay kit from Promega corporation, thereby reflecting the enzyme activity.
Experimental instrument:
An Echo650 pipetting system from Labcyte corporation; PERKIN ELMER company Envision microplate reader; eppendorf company 5810 centrifuge.
Experimental materials:
| Reagent(s) | Branding | Goods number |
| Tris hydrochloride solution | Sigma | T2663 |
| BRIJ 35detergent(10％) | Merck | 203728 |
| MgCl2 solution | Sigma | M1028 |
| ADP-Glo kinase detection kit | Promega | V9102 |
| JAK3 | Carna bioscience | 08-046 |
| Poly(4:1Glu,Tyr) | Sigma | P0275 |
| 384 Well plate | Perkin Elmer | 6007290 |
The experimental method comprises the following steps:
The test compounds were transferred to 384 well plates using an Echo pipetting system and 2 μl/well of JAK3 was added and incubated for 30 minutes. Then, 3. Mu.L/well of a mixed solution of substrate Poly (4:1 Glu, tyr) and ATP was added to initiate the enzyme reaction. The final concentrations of the compounds were 3-fold diluted in dimethyl sulfoxide (DMSO) starting from 300nM or 100nM, respectively. The final concentration of enzyme in the reaction was 1.9 ng/well, the final concentration of ATP was 36. Mu.M, and the final concentration of substrate was 0.1mg/mL. After 1 hour of reaction, 5. Mu.L/well ADP-GLO reagent was added and incubated for 40 minutes. Then 10. Mu.L/well kinase reaction detection reagent was added and incubated for 30 minutes. Fluorescence signals were read with an Envision microplate reader and the inhibition ratio, half inhibition concentration (IC50) was calculated.
The biological activity of the compounds of the invention was determined by the above assay and the IC50 values measured are given in table 2 below.
TABLE 2 IC for inhibition of JAK3 kinase activity by the compounds of the present invention50
| Examples compound numbering | IC50(nM) |
| 001 | 0.70 |
| 002 | 1.82 |
| 003 | 0.34 |
| 004 | 0.84 |
| 005 | 1.57 |
Test example 3: inhibition of BTK phosphorylation in Ramos cells
Experimental principle: after incubation of Ramos cells with the compound and the stimulator, fluorescence energy transfer was detected by Homogeneous Time Resolved Fluorescence (HTRF) method using the btsbio phosphorylation assay kit, reflecting the inhibition of phosphorylation.
Experimental instrument:
| Instrument for measuring and controlling the intensity of light | Branding | Model number |
| Echo | Labcyte | 650 |
| Biological safety cabinet | ESCO | CLASSⅡBSC |
| Centrifugal machine | Eppendorf | 5810 |
| CO2 incubator | ESCO | CCL-170B-8 |
| Cell counter | CountStar | IC1000 |
| Envision | Perkin Elmer | / |
Experimental materials:
The experimental method comprises the following steps:
The test compounds were transferred to 384 well plates using an Echo pipetting system, the Ramos cell density was adjusted to 1X107 cells/mL, 10 μl/well cell suspension was added and incubated for 1 hour in an incubator at 37 ℃ with 5% co2. Then 5. Mu.L/well of stimulator anti-human IgM antibody was added, the final stimulator concentration was 10. Mu.g/mL, and incubated for 10 minutes. The final concentration of the compound was 1. Mu.M, starting with 4-fold dilution in dimethyl sulfoxide (DMSO). mu.L/well of cell lysate was added and incubated for 30 min at room temperature. The extent of phosphorylation of BTK was detected using the Cisbio BTK phospho-Y223 kit, and finally the fluorescence signals at 665nm and 615nm were read on an Envision microplate reader to calculate the inhibition and half maximal inhibition concentration (IC50).
The biological activity of the compounds of the invention was determined by the above assay and the IC50 values determined are shown in Table 3 below.
TABLE 3 inhibitory Activity of the inventive Compounds against BTK phosphorylation in Ramos cells
| Examples compound numbering | IC50(nM) |
| 001 | 1.89 |
| 002 | 10.33 |
| 003 | 1.10 |
| 004 | 4.26 |
| 005 | 8.39 |
Test example 4: inhibition of STAT5 phosphorylation in CTLL-2 cells
Experimental principle: this experiment was performed to evaluate the effect of compounds on STAT5 phosphorylation of JAK3 downstream substrates. After incubation of CTLL-2 cells with the compound and the stimulator, fluorescence energy transfer between donor and acceptor microbeads was detected by time-resolved fluorescence using PERKIN ELMER company p-STAT5 (Tyr 694/699) detection kit, reflecting inhibition of phosphorylation.
Experimental instrument:
| Instrument for measuring and controlling the intensity of light | Branding | Model number |
| Biological safety cabinet | Thermo Scientific | 1300Series A2 |
| Centrifugal machine | Eppendorf | 5702 |
| CO2 incubator | Thermo Scientific | 371 |
| Cell counter | Invitrogen | C10281 |
| Envision | Perkin Elmer | / |
| Echo | Labcyte | 655 |
Experimental materials:
The experimental method comprises the following steps:
CTLL-2 cells were seeded in 384-well plates, 1.5X104 cells/15. Mu.l/well, compounds were transferred to 384-well plates with Echo and incubated for 30min in an incubator at 37℃with 5% CO2. Then 5. Mu.L/well of stimulator IL-2 was added at a final concentration of 1ng/mL and incubated for 30 minutes. The final concentration of the compound was 3-fold diluted in dimethyl sulfoxide (DMSO) starting from 3 μm. mu.L/well cell lysate was added and incubated at room temperature for 10 minutes. The phosphorylation degree of STAT5 was detected with PERKIN ELMER company ALPHALISAP-STAT5 (Tyr 694/699) detection kit, and finally the AlphaLISA signal was read on an Envision microplate reader to calculate the inhibition and half inhibition concentration (IC50).
The biological activity of the compounds of the invention was determined by the above assay and the IC50 values measured are shown in Table 4 below.
TABLE 4 inhibitory Activity of the inventive Compounds against STAT5 phosphorylation in CTLL-2 cells
| Examples compound numbering | IC50(nM) |
| 001 | 5.70 |
| 002 | 34.63 |
| 003 | 20.96 |
| 005 | 12.23 |