CN101516893B - Novel 5, 7-disubstituted [1, 3 ] thiazolo [4, 5-d] pyrimidin-2 (3h)-one derivatives and their use in therapy - Google Patents

Novel 5, 7-disubstituted [1, 3 ] thiazolo [4, 5-d] pyrimidin-2 (3h)-one derivatives and their use in therapy Download PDF

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CN101516893B
CN101516893B CN2007800360541A CN200780036054A CN101516893B CN 101516893 B CN101516893 B CN 101516893B CN 2007800360541 A CN2007800360541 A CN 2007800360541A CN 200780036054 A CN200780036054 A CN 200780036054A CN 101516893 B CN101516893 B CN 101516893B
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amino
thiazole
ethyl
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CN101516893A (en
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罗尔夫·约翰森
索菲娅·卡尔斯特罗姆
安妮卡·克斯
冈纳·诺德瓦尔
托拜厄斯·雷恩
坎·斯利沃
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Cancela Joint Stock Company
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AstraZeneca AB
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Abstract

The present invention discloses novel 5,7-disubstituted [1,3]thiazolo[4,5-d]pyrimidin-2(3H)-one derivatives of formula (I) wherein R<1>, R<2>, R<3>, R<4> and n are as defined in the specification, and pharmaceutically acceptable salts thereof, together with processes for their preparation, pharmaceutical compositions comprising them and their use in therapy. The compounds of formula (I) are CX3CR1 receptor antagonists and are thereby particularly useful in the treatment or prophylaxis of neurodegenerative disorders, demyelinating disease, cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, rheumatoid arthritis, pulmonary diseases such as COPD, asthma or pain.

Description

5,7-dibasic [1,3] thiazole is [4,5-D] pyrimidines-2 (3H)-ketone derivatives and their purposes in treatment also
Technical field
The invention discloses 5 of novelty, 7-dibasic [1,3] thiazole is [4,5-d] pyrimidines-2 (3H)-ketone derivatives and preparation method thereof also, contains their pharmaceutical preparation and their purposes in treatment.
Background technology
Chemokine (Chemokines) plays an important role in the immunity of various diseases and illness and Inflammatory response, these diseases and illness comprise asthma (asthma), atherosclerosis (atherosclerosis) and allergic disease (allergic diseases), and autoimmunization pathology such as rheumatoid arthritis and multiple sclerosis (multiple sclerosis).These are belonged to ever-increasing 8-14kDa superfamily protein by oozy small molecules, and this family is characterized as conservative halfcystine motif.At present, the chemokine superfamily comprises the four class families that demonstrate the characteristic structural motif, i.e. C-X-C, C-C and C-X 3-C and XC family.C-X-C and C-C family have sequence similarity and be according to cysteine residues NH-near-end between single amino acids insert and distinguish mutually.C-X 3-C family be according to cysteine residues NH-near-end between three aminoacid insertion come to distinguish with other two families.On the contrary, the member of XC family lacks in preceding two cysteine residues one.
The C-X-C chemokine comprises several potent chemoattractant and the activator of neutrophilic granulocyte, is for example interleukin 8 (IL-8) and neutrophil activation peptide 2 (NAP-2).
The C-C chemokine comprises the potent chemoattractant of monocyte, lymphocyte and neutrophilic granulocyte.Example comprises person monocytic cell's chemotactic protein 1-3 (MCP-1, MCP-2 and MCP-3), RANTES (regulating active, normal T cell expressing and secretion), eotaxin (eotaxin) and macrophage inflammatory protein 1 α and 1 β (MIP-1 α and MIP-1 β).
C-X 3-C chemokine (also being called CXXXC chemotactic molecule (fractalkine)) is the microglia (microglia) in the central nervous system and the potent chemoattractant and the activator of monocyte, T cell, NK cell and mastocyte.
The effect that studies show that chemokine has in these acceptors to be called following acceptor: CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (C-C family) by G albumen-coupled receptor subfamily mediation; CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 (for C-X-C family) and CX3CR1 are (for C-X 3-C family).Can be used for treating for example above-mentioned those diseases and the illness of mentioning owing to regulate the medicine of these acceptors, so these acceptors are represented the excellent drug development goal.
WO 01/25242 discloses also [4,5-d] pyrimidine derivatives of some thiazole, and it is used as the antagonist of the acceptor that links to each other with C-C chemokine family with C-X-C, in particular as the CXCR2 receptor antagonist.
The present invention relates to a compounds relevant, but its structure type is specifically not illustrational in WO 01/25242 with disclosed compound among the WO 01/25242.When comparing with disclosed embodiment among the WO 01/58907, The compounds of this invention is as CX 3The antagonist of CR1 acceptor has shown beat all useful quality.
Summary of the invention
The invention provides the solvate of formula (I) compound or pharmaceutically acceptable salt thereof, solvate or the salt of free alkali form,
Figure G2007800360541D00021
Wherein:
R 1Expression CH 3Or CF 3
R 2Expression halogen, CN or C 1-6Alkyl;
R 3Expression H or CH 3
R 4Expression H or CH 3
N represents 0,1 or 2.
In one embodiment of the invention, it provides formula (I) compound, and wherein n represents 1.
In another embodiment of the present invention, it provides formula (I) compound, wherein R 1Expression CH 3
In another embodiment of the present invention, it provides formula (I) compound, wherein R 2Expression halogen or CN.
In another embodiment of the present invention, it provides formula (I) compound, wherein R 2Expression F or Cl.
In another embodiment of the present invention, it provides formula (I) compound, wherein R 2Expression CN.
In another embodiment of the present invention, it provides formula (I) compound, and wherein n represents 1; R 1Expression CH 3, and R 2Expression F, Cl or CN.
In another embodiment of the present invention, it provides formula (I) compound, and wherein pyridine connects with its 5 and has Cl 2 of pyridine.
In another embodiment of the present invention, it provides formula (I) compound, and wherein pyridine connects with its 2 and has CN 4 of pyridine.
In another embodiment of the present invention, it provides formula (I) compound, and wherein pyridine connects with its 2 and has F 5 of pyridine.
In another embodiment of the present invention, it provides formula (I) compound, and wherein pyridine connects with its 2 and has Cl 5 of pyridine.
In another embodiment of the present invention, it provides formula (I) compound, and wherein pyridine connects with its 2 and has F 3 of pyridine.
In another embodiment of the present invention, it provides formula (I) compound, and wherein pyridine connects with its 4 and has F 3 of pyridine.
In another embodiment of the present invention, it provides formula (I) compound, wherein R 3Expression H.
In another embodiment of the present invention, it provides formula (I) compound, wherein R 4Expression CH 3
In another embodiment of the present invention, it provides the solvate of formula (I) compound or pharmaceutically acceptable salt thereof, solvate or the salt of free alkali form, and described compound is selected from:
5-{[(1S)-and 1-(5-chloropyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1R)-and 1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
2-{ (1S)-1-[(7-{[(1R)-1-(methylol)-3-methyl butyl] amino }-2-oxo-2,3-dihydro [1,3] thiazole is [4,5-d] pyrimidine-5-yl also) sulfenyl] ethyl } pyridine-4-formonitrile HCN;
5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol) butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also; And
5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-[[(1R)-and 1-(methylol) butyl] (methyl) amino] [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also.
Formula (I) compound can exist with stereoisomeric forms in any ratio and/or tautomeric form.Should be appreciated that all enantiomers, diastereomer, racemic modification, tautomer and composition thereof all comprise within the scope of the invention.
When comparing with disclosed compound among the WO 01/25242, The compounds of this invention is characterised in that 5 sulfenyl Alkylpyridyls that have branching in thiazole and pyrimidine ring system.In other words, to have introduced be not the R of hydrogen to The compounds of this invention 1Group.
According to the present invention, it also provides the method for preparation formula (I) compound or pharmaceutically acceptable salt thereof, and described method comprises:
A) make the reaction of formula (II) compound and formula (III) compound,
Formula (II) compound is:
Figure G2007800360541D00041
R wherein 3And R 4Suc as formula defining in (I);
Formula (III) compound is:
Figure G2007800360541D00051
R wherein 1, R 2Define suc as formula (I) is middle with n, and L 1The expression leaving group; Or
B) make formula (IV) compound hydrolysis,
Figure G2007800360541D00052
R wherein 1, R 2, R 3, R 4Define suc as formula (I) is middle with n;
And in case of necessity, formula (I) compound or its another kind of salt of gained is converted into pharmacologically acceptable salt; Maybe formula (I) compound that obtains is converted into another compound of formula (I) compound; And when needing, formula (I) compound that forms is converted into its optical isomer.
In method (a), make reactant (II) and (III) in appropriate organic solvent such as dimethyl sulfoxide (DMSO) (DMSO), acetonitrile or 1-Methyl-2-Pyrrolidone (NMP), be coupled at together.Described reaction is chosen wantonly at the organic bases that adds or mineral alkali such as triethylamine, N, and N-diisopropylethylamine (DIPEA) or sodium hydride carry out under existing.Described being reflected under gentle reductive agent such as the sodium borohydride existence carried out.Describedly be reflected at suitable temperature and carry out, the temperature between the boiling point of room temperature and solvent is carried out usually.Described reaction continues about one hour for some time to a week usually, or finishes up to analyzing the formation that shows required product.Suitable leaving group L 1Be halogen, particularly chlorine or bromine.In one embodiment, L 1Expression chlorine.
In method (b), make reactant (IV) in appropriate organic solvent as 1, stand acid catalyzed hydrolysis in 4-dioxane, tetrahydrofuran (THF) (THF), dimethyl sulfoxide (DMSO) (DMSO) or the 1-Methyl-2-Pyrrolidone (NMP).Suitable acid comprises mineral acid example hydrochloric acid or Hydrogen bromide, or organic acid such as trifluoroacetic acid.Describedly be reflected at suitable temperature, the temperature between the boiling point of room temperature and solvent is carried out usually.Described reaction continues about one hour to one day for some time usually, or finishes up to analyzing the formation that shows required product.
Obvious to those skilled in the art is, may be expectation or necessary to amine, hydroxyl or other potential reactive group protection in aforesaid method.Suitable protecting group and interpolation or to remove the details of method of described group normally well known in the art.Referring to, " the Protective Groups in Organic Synthesis " that for example Greene and Wuts showed, 3rd Edition (1999).
The present invention includes formula (I) compound of salt form.Suitable salt comprises the salt with organic acid or mineral acid or organic bases or mineral alkali formation.Described salt will be pharmacologically acceptable salt usually, but can utilize the salt of non-pharmaceutically acceptable acid or alkali when preparation and the described compound of purifying.
The salt of formula (I) compound can followingly form: make free compound or its salt, enantiomer or racemic modification and one or how normal suitable acid or alkali reaction.Described reaction can described salt be insoluble to wherein solvent or medium in carry out, or described salt be dissolved in wherein solvent (for example, water, dioxane, ethanol, tetrahydrofuran (THF) or ether, or the mixture of solvent) in carry out, but these solvent vacuum are removed or are removed by lyophilize.Described reaction also can be metathesis process (metatheticalprocess), or it can carry out on ion exchange resin.
Formula (II) compound can use to those skilled in the art obvious easily currently known methods preparation usually.A kind of such appropriate route is shown in the scheme 1.
Scheme 1
Figure G2007800360541D00061
Formula (III) compound is commercially available, or known in the document, maybe can use to those skilled in the art obvious easily currently known methods preparation.
Formula (IV) compound is known from for example WO 01/25242 or WO 05/33115, maybe can use to those skilled in the art obvious easily currently known methods preparation.A kind of such synthetic route is shown in the scheme 2.
Scheme 2
Figure G2007800360541D00071
The formula V compound is known from WO 01/58907, WO 01/25242 or WO 02/76990, maybe can use to those skilled in the art obvious easily currently known methods preparation.
For example, formula V compound and formula (VI) compound that obtains thus can prepare shown in scheme 3:
Scheme 3
Preparation formula (II), (III), (IV) and (V) the suitable concrete grammar of compound be described in detail in the embodiment part of this specification sheets, and described method has been represented the specific embodiments of the inventive method.
Midbody compound can use same as before or use with shielded form.Suitable protecting group and interpolation or to remove the details of method of described group normally well known in the art.Referring to, " the Protective Groups in Organic Synthesis " that for example Greene and Wuts showed, 3rd Edition (1999).
The compounds of this invention and the intermediate that obtains described compound thereof can separate from their reaction mixture, and in case of necessity by using standard technique to be further purified.
Formula (I) compound can exist with stereoisomer form.Therefore, all enantiomer, diastereomer, racemic modification and composition thereof all comprise within the scope of the invention.Various optical isomers can be separated by the stereoisomer mixture that adopts routine techniques (for example fractional crystallization or HPLC) separating compound.
Perhaps, various optical isomers can be directly with the preparation of optics activated feedstock.
Formula (I) compound contains two stereocenters, thus can be to exist suc as formula the different stereoisomeric forms in any ratio of four kinds shown in (Ia) to (Id).
Figure G2007800360541D00081
All these four kinds of steric isomers and any mixture thereof all comprise within the scope of the invention.In one embodiment, formula (I) compound has the stereochemistry shown in the formula (Ia).In another embodiment, formula (I) compound has the stereochemistry shown in the formula (Ib).
Midbody compound also can exist with stereoisomeric forms in any ratio, and can use with the form of enantiomer, diastereomer, racemic modification or the mixture of purifying.
In this manual, term " C 1-6Alkyl " comprise straight chain and branched-chain alkyl and cycloalkyl.Has 1 to 6 carbon atom C 1-6Alkyl, described alkyl can be but be not limited to: methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl, n-pentyl, isopentyl, tert-pentyl, neo-pentyl, n-hexyl, isohexyl or cyclohexyl.
In this manual, term " halo (halo) " or " halogen (halogen) " are meant fluorine, chlorine, bromine and iodine.
Formula (I) compound and pharmacologically acceptable salt thereof are useful, because they have as CX 3The pharmacological activity of CR1 receptor antagonist.Particularly, when comparing with concrete illustrational compound among the WO 01/25242, formula of the present invention (I) compound has the inhibition CX of remarkable improvement 3The effectiveness of the inhibition CXCR2 acceptor of the effectiveness of CR1 acceptor and/or reduction.Preferred The compounds of this invention demonstrates enhanced simultaneously and suppresses CX 3The effectiveness of the inhibition CXCR2 of the effectiveness of CR1 and reduction.
In one aspect, the invention provides formula (I) compound or pharmaceutically acceptable salt thereof, it is as medicine.
On the other hand, the invention provides formula (I) compound or pharmaceutically acceptable salt thereof is used for the treatment of or prevents CX in preparation 3The CR1 acceptor carries out the purposes in the medicine that antagonism is useful disease or illness.
On the other hand, the invention provides formula (I) compound or pharmaceutically acceptable salt thereof is used for the treatment of or prevents purposes in the medicine of neurodegenerative disease (neurodegenerative disorder), demyelinating disease (demyelinatingdisease), cardiovascular and cerebrovascular atheromatosis (cardio-and cerebrovascularatherosclerotic disorder), peripheral arterial disease, rheumatoid arthritis, tuberculosis disease such as COPD (chronic obstructive pulmonary disease), asthma or pain in preparation.
On the other hand, the invention provides formula (I) compound or pharmaceutically acceptable salt thereof is used for the treatment of or prevents purposes in the medicine of multiple sclerosis (MS) in preparation.
On the other hand, the invention provides the purposes of formula (I) compound or pharmaceutically acceptable salt thereof in the medicine that preparation is used for the treatment of or prevention of arterial is atherosis, described medicine by prevent and/or reduce new atherosclerotic lesions or patch formation and/or by the progress that prevents or slow down existing damage and patch treat or prevention of arterial atherosis.
On the other hand, the invention provides the purposes of formula (I) compound or pharmaceutically acceptable salt thereof in the medicine that preparation is used for the treatment of or prevention of arterial is atherosis, described medicine treat with the danger that reduces plaque rupture and atherosclerosis thrombosis incident by the composition that changes patch or prevention of arterial atherosis.
On the other hand, the invention provides formula (I) compound or pharmaceutically acceptable salt thereof is used for the treatment of or preventing apoplectic or transience brain injury (transient brain injury, the purposes in medicine TBI) in preparation.
According to the present invention, it also provides a kind of treatment to CX 3CR1 carries out antagonism to be useful disease or illness or to reduce CX 3It is the useful disease or the method for illness danger that CR1 carries out antagonism, described method comprise will the treatment significant quantity formula (I) compound or pharmaceutically acceptable salt thereof be administered to and suffer from described disease or illness or face described disease or the people of illness danger.
The present invention also provides a kind of and is suffering from neurodegenerative disease, demyelinating disease, the cardiovascular and cerebrovascular atheromatosis, peripheral arterial disease, rheumatoid arthritis, tuberculosis disease such as COPD, the people of asthma or pain or face neurodegenerative disease, demyelinating disease, the cardiovascular and cerebrovascular atheromatosis, peripheral arterial disease, rheumatoid arthritis, tuberculosis disease such as COPD, the philtrum of asthma or pain danger, treat described disease or illness or reduce described disease or the method for illness danger, wherein said method comprises that formula (I) compound or pharmaceutically acceptable salt thereof with the treatment significant quantity is administered to described people.
The present invention also is provided to suffer from multiple sclerosis (MS) or face the dangerous philtrum of multiple sclerosis (MS) and treats described disease or illness or reduce described disease or the method for illness danger, and wherein said method comprises that formula (I) compound or pharmaceutically acceptable salt thereof with the treatment significant quantity is administered to the people.
The present invention also is provided to be suffered from multiple sclerosis (MS) or faces the dangerous philtrum of multiple sclerosis (MS), by preventing and/or reduce the formation of new atherosclerotic lesions or patch and/or treat or reduce the method for described disease or illness danger that wherein said method comprises that formula (I) compound or pharmaceutically acceptable salt thereof that will treat significant quantity is administered to the people by the progress that prevents or slow down existing damage and patch.
The present invention also is provided at the philtrum of suffering from atherosclerosis or facing atherosclerosis danger, composition by changing patch is to reduce the danger of plaque rupture and atherosclerosis thrombosis incident, treat or reduce the method for described disease or illness danger, wherein said method comprises that formula (I) compound or pharmaceutically acceptable salt thereof with the treatment significant quantity is administered to the people.
On the other hand, the invention provides a kind of pharmaceutical preparation, it comprises formula (I) compound or pharmaceutically acceptable salt thereof for the treatment of significant quantity, and is mixed with pharmaceutically acceptable adjuvant, diluent or carrier, and this pharmaceutical preparation is used for the treatment of or prevents CX 3The antagonism of CR1 acceptor is useful disease or illness.
On the other hand, the invention provides a kind of pharmaceutical preparation, it comprises formula (I) compound or pharmaceutically acceptable salt thereof for the treatment of significant quantity, and being mixed with pharmaceutically acceptable adjuvant, diluent or carrier, this pharmaceutical preparation is used for the treatment of or prevents neurodegenerative disease, demyelinating disease, cardiovascular and cerebrovascular atheromatosis, peripheral arterial disease, rheumatoid arthritis, COPD, asthma or pain.
On the other hand, the invention provides a kind of pharmaceutical preparation, it comprises formula (I) compound or pharmaceutically acceptable salt thereof for the treatment of significant quantity, and is mixed with pharmaceutically acceptable adjuvant, diluent or carrier, and this pharmaceutical preparation is used for the treatment of or prevents multiple sclerosis.
On the other hand, the invention provides a kind of pharmaceutical preparation, it comprises formula (I) compound or pharmaceutically acceptable salt thereof for the treatment of significant quantity, and be mixed with pharmaceutically acceptable adjuvant, diluent or carrier, this pharmaceutical preparation by prevent and reduce new atherosclerotic damage and/or patch formation and/or by the progress that prevents or slow down existing damage and patch treat or prevention of arterial atherosis.
Described compound can be used as monotherapy and uses, or as the inflammatory conditions of central nervous system and the preventative or therapeutic treatment of disease (as apoplexy and transience brain injury (TBI)) are used (Soriano et al.J.Neuroimmunology 2002 with associated form, 125,59-65.).
On the other hand, the invention provides a kind of pharmaceutical preparation, it comprises formula (I) compound or pharmaceutically acceptable salt thereof for the treatment of significant quantity, and be mixed with pharmaceutically acceptable adjuvant, diluent or carrier, this pharmaceutical preparation treat with the danger that reduces plaque rupture and atherosclerosis thrombosis incident by the composition that changes patch or prevention of arterial atherosis.
Formula (I) compound and pharmacologically acceptable salt thereof are intended to be used for the treatment of or prevent CX 3It is the disease or the illness of expectation that the activity of CR1 acceptor is regulated.Particularly, described compound is intended to be used in Mammals (comprising the people) treatment neurodegenerative disease or demyelinating disease.More specifically, described compound is intended to be used for the treatment of multiple sclerosis.Described compound also is intended to be used for the treatment of pain, rheumatoid arthritis, osteoarthritis, cardiovascular and cerebrovascular atheromatosis, peripheral arterial disease and pulmonary hypertension (pulmonary arterial hypertension).
The illness that can specifically mention is: neurodegenerative disease and dementia disease, for example, alzheimer's disease (Alzheimer ' s Disease), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis) and other motor neurone disease, Ke-Ya syndrome (Creutzfeld-Jacob Disease) and other protein virus disease, HIV encephalopathic (HIV encephalopathy), Huntington Chorea (Huntington ' s Disease), frontotemporal dementia (Frontotemporal dementia), Lu Yi body dementia (Lewy body dementia) and vascular dementia (vascular dementia); Polyneuropathy (polyneuropathies), for example Ji-Ba syndrome (Guillain-Barr é syndrome), chronic inflammatory demyelinating polyneuropathy (Chronic InflammatoryDemyelinating Polyneuropathy), multifocal motor neuropathy (multifocal motorneuropathy) and plexopathy (plexopathies); The CNS demyelination, for example, acute dispersivity/hemorrhagic encephalomyelitis (acute disseminated/haemorrhagic encephalomyelitis) and subacute sclerosing panencephalitis (subacute sclerosing panencephalitis); Neuromuscular disease (neuromuscular disorders), for example myasthenia gravis (myasthenia gravis) and Lan-Yi syndrome (Lambert-Eaton syndrome); Spinal disease (spinal disorders), for example tropical spastic paraparesis (tropical spastic paraparesis) and stiff man syndrome (stiff-man syndrome); Paraneoplastic syndrome (paraneoplastic syndromes), for example cerebellar degeneration (cerebellar degeneration) and encephalomyelitis (encephalomyelitis); Traumatic brain injury (traumatic brain injury); Migraine; Cancer; Allograft rejection (allograft rejection); Sjogren's syndrome (systemic sclerosis); Virus infection; Parasite spread disease (parasite-transmitted disease), for example malaria; Periodontopathy (periodontal disease); Myocardial infarction (myocardial infarction); Apoplexy; Coronary heart disease (coronaryheart disease), ischemic heart disease (ischaemic heart disease); And restenosis (restenosis); Rheumatoid arthritis; Tuberculosis disease such as COPD; Asthma or pain.
The compounds of this invention also is intended to by preventing and/or reducing the formation of new atherosclerotic damage and/or patch and/or be used for the treatment of atherosclerosis by the progress that prevents or slow down existing damage and patch.
Thereby The compounds of this invention also is intended to be used for the treatment of anti-atherosclerosis by the composition reduction plaque rupture that changes patch and the danger of atherosclerosis thrombosis incident.
The compounds of this invention also is intended to by the alleviation of inducing IBD and/or keeps alleviation, thereby is used for the treatment of inflammatory bowel disease (IBD), for example Crohn's disease (Crohn ' s disease) and ulcerative colitis.
Prevention (prophylaxis) is considered to the people who treats the previous outbreak of suffering from described disease or illness or to be considered to face the people of described disease of suffering from of increase or illness danger relevant especially.Face the people who develops into disease specific or illness danger generally include have described disease or illness family history or by genetic test or Screening and Identification for developing into the people of described disease or illness especially easily.
For above-mentioned treatment indication, the dosage of administration will change with employed compound, administering mode and required treatment certainly.Yet, usually when compound with every day 1mg-2000mg the solid form dosed administration time, just obtain gratifying result.
Formula (I) compound and pharmaceutically acceptable derivative thereof can use separately or use with the form of suitable pharmaceutical composition, and in composition, described compound or derivative mix with pharmaceutically acceptable adjuvant, vehicle or carrier.Administration can be passed through, but is not limited to, in intestines (comprise per os, through hypogloeeis or per rectum), intranasal, intravenously, part or other parenteral route.Select and the routine operation of preparation suitable pharmaceutical formulation is described in, for example " Pharmaceuticals-The Science of Dosage Form Designs ", M.E.Aulton, Churchill Livingstone is in 1988.Pharmaceutical composition preferably contains and is lower than 80%, the formula more preferably less than 50% (I) compound or pharmaceutically acceptable salt thereof.
The present invention also provides the method for preparing this pharmaceutical composition, and it comprises each composition of mixing.
The invention still further relates to combination therapy, wherein when formula (I) compound or pharmaceutically acceptable salt thereof comprise the pharmaceutical composition of formula (I) compound or preparation in being used for the treatment of cardiovascular and cerebrovascular atheromatosis and peripheral arterial disease each treatment and/or medicine simultaneously or administration successively.
Particularly, formula (I) compound or pharmaceutically acceptable salt thereof can be with one or more compound administrations of following group:
1) antiphlogiston, for example
A) NSAID (for example acetylsalicylic acid, Ibuprofen BP/EP, Naproxen Base, flurbiprofen, diclofenac and indomethacin);
B) the leukotrienes synthetic inhibitor (the 5-LO inhibitor, for example AZD4407, abandon and stay logical, licofelone, CJ13610 and CJ13454; FLAP inhibitor, for example BAY-Y-1015, DG-031, MK591, MK886 and A81834; LTA4 hydrolase inhibitor, for example SC56938 and SC57461A);
C) leukotrienes receptor antagonist (for example, CP195543, A Meiluban (amelubant), LY293111, Zafirlukast (accolade) and MK571);
2) antihypertensive drug, for example
A) beta-Blocking agent (for example metoprolol, atenolol USP 23 and sotalol);
B) angiotensin-convertion enzyme inhibitor (for example captopril, Ramipril, quinapril and enalapril);
C) calcium channel blocker (for example verapamil, Odizem, felodipine and amlodipine);
D) angiotensin II receptor antagonists (for example irbesartan, Candesartan, telmisartan (telemisartan) and losartan);
3) anti-coagulant, for example
A) thrombin inhibitors (for example Melagatran (ximelagatran)), heparin and coagulation factor xa inhibitors;
B) anticoagulant (for example clopidogrel (clopidrogrel), ticlopidine, prasugel and AZ4160);
4) lipid metabolism conditioning agent, for example
A) insulin sensitizer such as PPAR agonist (for example U-721017E, rosiglitazone, Galida, muraglitazaar, gefemrozil and fenofibrate);
B) HMG-CoA reductase inhibitor, Statins (for example Simvastatin, Pravastatin, atorvastatin, superstatin, fluvastatin and pitavastatin);
C) cholesterol absorption inhibitor (for example ezetimibe);
D) ibat inhibitor (for example, AZD-7806);
E) lxr agonist (for example, GW-683965A and T-0901317);
F) FXR receptor modulators;
G) inhibitor of phospholipase enzymes;
5) anti-anginal drug, for example, nitrate and nitrite;
6) oxidative stress conditioning agent, for example, antioxidant (probucol) and myeloperoxidase inhibitor.
Concrete embodiment
The present invention will be described by the following example, but the present invention is not limited to these embodiment:
General method
All solvents that use all are AGs, and commercially available anhydrous solvent is that reaction is conventional employed.Reaction is carried out in the inert atmosphere of nitrogen or argon gas usually.
Use to be equipped with the 5mm BBO probe of Z gradient Varian Unity+400NMR spectrograph, be equipped with the anti-Bruker DPX400 NMR spectrograph record 400MHz (proton) that flows the Bruker Avance 400 NMR spectrographs of probe or be equipped with the 4-nuclear probe of Z gradient of 60 μ l binary of Z gradient 1H NMR spectrum and 100MHz's (carbon-13) 13C NMR spectrum.On the Bruker av600 NMR spectrograph of the 5mm BBI probe that is equipped with the Z gradient, write down 600MHz 1H NMR spectrum.On Varian Gemini 300 NMR that are equipped with 5mm BBI probe, write down 300MHz 1H NMR spectrum.On VarianInova 500 spectrographs of 11.74T magnetic field operation, write down 500MHz at outfit 5mm nuclear gradient probe (nuclei gradient probe) 1H NMR spectrum.Unless specify record 400MHz (proton) spectrum and 100MHz (carbon-13) spectrum in an embodiment.Use following reference signal: DMSO-d 6Center line δ 2.50 ( 1H), δ 39.51 ( 13C); CD 3OD center line δ 3.31 ( 1H) or δ 49.15 ( 13C); Acetone-d 62.04 ( 1H), 206.5 ( 13C); And CDCl 3δ 7.26 ( 1H), CDCl 3Center line δ 77.16 ( 13C) (except as otherwise noted).
Upward or at Cyclosil B post (thermograde 110-130 ℃) upward determine enantiomeric excess (ee) at Cyclodex B post (100 ℃ of constant temperature wash-outs) by GC.Determine diastereomeric excess (de) by HPLC.
Record mass spectrum on the Waters LCMS that forms by Alliance 2795 (LC) and the single quadrupole mass spectrometer of ZQ.Mass spectrograph is equipped with the electric spray ion source (ESI) with positive ion mode or negative ion mode operation.Capillary voltage is 3kV, and mass spectrograph scans between m/z 100-700, and be 0.3s or 0.8s sweep time.At X-Terra MS, separate on C8-post (3.5 μ m, 50 or 100mm * 2.1mm internal diameter) or the ScantecLab ' s ACE 3 AQ posts (100mm * 2.1mm internal diameter).Column temperature is set at 40 ℃.Use linear gradient, use neutral or acid flow phase system, the organic phase with 0% to 100% in 4-5 minute is moved flow velocity 0.3ml/min.Neutral flow phase system: acetonitrile/[10mMNH 4OAc (aqueous solution)/MeCN (95: 5)] or [10mM NH 4OAc (aqueous solution)/MeCN (1/9)]/[10mM NH 4OAc (aqueous solution)/MeCN (9/1)].Acid flow phase system: [133mM HCOOH (aqueous solution)/MeCN (5/95)]/[8mM HCOOH (aqueous solution)/MeCN (98/2)].
Perhaps, on the MicromassLCT mass spectrograph that is equipped with the electric spray ion source of operating with positive ion mode (ESI), write down mass spectrum.
On the GC-MS that provides by Agilent Technologies (GC 6890,5973N MSD), carry out compound identification.The post that uses is VF-5 MS ID 0.25mm * 30m, 0.25 μ m (Varian Inc.).Use the linear temperature gradient, originate in 40 ℃ (keeping 1 minute), end at 300 ℃ (keeping 1 minute), 25 ℃/minute.MS is equipped with the EI ion source.MS scans between m/z 50-500, and sweep velocity was made as for 3.25 scanning/seconds.Electronic voltage is made as 70eV.
Use and carry out the HPLC analysis in the Agilent HP1000 system that forms by G1379A micro-vacuum degasser, G1312A binary pump, G1367A orifice plate automatic sampler, G1316A constant temperature column compartment and G1315B diode-array detector.Post: X-Terra MS, Waters, 4.6 * 50mm, 3.5 μ m.Column temperature is set at 40 ℃, flow velocity 1.5ml/ minute.Diode-array detector scans from 210-300nm, and step-length and peak width are made as 2nm and 0.05 minute respectively.Use linear gradient, in 4 minutes, move to 100% acetonitrile from 0%.Moving phase: 5% acetonitrile solution that in MilliQ Water, has acetonitrile/10nM ammonium acetate.
Typical reaction postprocessing method (typical workup procedure after a reaction) comprising: with solvent (for example ethyl acetate) extraction product; Wash with water, use MgSO subsequently 4Or Na 2SO 4Dry organic phase is carried out vacuum concentration to solution then.
At Merck TLC-plate (silica gel 60F 254) on carry out thin-layer chromatography (TLC), spot is manifested by UV.At Combi CompanionTM (using the quick post of RediSepTM positive) goes up or carry out flash chromatography on Merck silica gel 60 (0.040-0.063mm).The typical solvent of flash column chromatography is the mixture of chloroform/methanol, the mixture of toluene/ethyl acetate and the mixture of ethyl acetate/hexane.
Unless explanation is arranged in an embodiment in addition, in the Gilson that is equipped with diode-array detector being prepared property chromatogram on the preparation property HPLC automatically, use XTerra MS post (C8,19 * 300mm, 7 μ m), in MilliQ Water, has the gradient of 5% acetonitrile solution of acetonitrile/0.1M ammonium acetate, in 13 minutes, move to 60% acetonitrile from 20%, and flow velocity is 20ml/ minute.Perhaps, purifying is partly carrying out on the preparation property Shimadzu LC-8A HPLC, and it is furnished with Waters
Figure G2007800360541D00152
Post (C18,5 μ m, the Shimadzu SPD-10A UV-vis.-detector of 100mm * 19mm).Gradient is acetonitrile/0.1% trifluoroacetic acid in MilliQWater, moves to 60% acetonitrile from 35% in 20 minutes.Flow velocity: 10ml/ minute.Perhaps being prepared property HPLC on Agilent 1100 instruments that UV detects is being housed.Post: Kromasil-C18,20 * 250mm, 10 μ m.Carry out isocratic elution with moving phase acetonitrile/MilliQ Water/ formic acid (46/54/0.1).Flow velocity: 19ml/min.
Usually in the mixture (as ether, ethyl acetate/heptane and methanol) of solvent or solvent, carry out recrystallization.
Use following abbreviation:
The DCM=methylene dichloride;
The de=diastereomeric excess;
DIPCl=β-chlorine diisopinocampheylchloroborane thiazolinyl borine (β-chlorodiisopinocamphenylborane, DIP-Chloride TM);
DIPEA=N, the N-diisopropylethylamine;
DMF=N, dinethylformamide;
The DMSO=dimethyl sulfoxide (DMSO);
The ee=enantiomeric excess;
The NCS=N-chloro-succinimide;
The NMP=1-N-methyl-2-2-pyrrolidone N-;
The THF=tetrahydrofuran (THF);
The aq=aqueous solution; With
Conc=is spissated.
Used raw material is that commercial source obtains or according to document operation preparation, and has and report consistent experimental data.
The example of prepared raw material is as follows:
(2R)-and 2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol: WO 02/076990 (embodiment 1-4);
5-(benzyl sulfenyl)-7-chlorine [1,3] thiazole is [4,5-d] pyrimidine-2-amine: WO 00/09511 (embodiment 6 and 7) also;
5-fluoro-pyridine-2-formonitrile HCN: WO 2005/066155 (embodiment 2);
1-(3-fluorine pyridin-4-yl) ethanol: Marsais, F.et al.Tetrahedron 1983,39,2009-2021 (embodiment 3);
2-ethanoyl-pyridine-4-formonitrile HCN: Citterio et al.J.Chem.Res.Synopses 1982,10,272-273 (embodiment 5);
1-(6-chloropyridine-3-yl) ethyl ketone: Lee, C.et al.J.Med.Chem.2001,44,2133 (embodiment 6 and 7).
In general method subsequently, R 3And R 4Suc as formula defining in (I); Py represents optional substituted pyridyl, and LG represents leaving group.
General method A
In nitrogen atmosphere, sodium borohydride (0.1 equivalent), DIPEA (1.5 equivalent) and compound (III) (1.2 equivalent) are added among compound (V) (1.0 equivalent)/DMSO.The reaction mixture that forms is finished (by LC-MS, HPLC or TLC monitoring) 40 ℃ of stirrings up to reaction.Mixture is poured in the frozen water, then product is extracted with DCM or EtOAc.With the organic phase drying that merges, vacuum concentration then.In case of necessity, use prepares property HPLC or through flash column chromatography crude product is carried out purifying.
General method B
Figure G2007800360541D00172
Dense HCl (2.5mL/mmol compound (VI)) is added to compound (VI) (1.0 equivalent)/CH 3Among the CN.Reaction mixture is cooled off in ice bath, drip the Sodium Nitrite (2.0 equivalent) that is dissolved in the minimum water gaging then.Reaction mixture is finished (by LC-MS, HPLC or TLC monitoring) 0 ℃ of stirring up to reaction, pour into then in the frozen water, with the sodium bicarbonate neutralization, then with DCM or EtOAc extraction.With the organic phase drying that merges, vacuum concentration obtains product then.
General method C
The potassium hydroxide (2.0 equivalent) that will be dissolved in the methyl alcohol is added drop-wise in compound (VII) (1.0 equivalent) cold (0 ℃) solution in methyl alcohol.The mixture that forms is finished (by LC-MS, HPLC or TLC monitoring) 0 ℃ of stirring up to reaction.Steam solvent, product just is used for next step without being further purified.
General method D
Figure G2007800360541D00182
Dense HCl (1.0 equivalent) solution is added to compound (IV) (1.0 equivalent) in 1, in cold (0 ℃) solution in the 4-dioxane.The mixture that forms 40 ℃ of stirrings, is finished (by LC-MS, HPLC or TLC monitoring) up to reaction.With reaction mixture NaHCO 3(aqueous solution) neutralization steams dioxane then.Resistates is dissolved among DCM or the EtOAc, uses the salt water washing, dry and vacuum concentration.In case of necessity, use prepares property HPLC or through flash column chromatography crude product is carried out purifying.
General method E1
Figure G2007800360541D00183
At 0 ℃ with in nitrogen atmosphere, the solution of compound (VIII) (1.0 equivalent) in THF is added to (obtains compound (IX)) among (+)-DIPCl or be added among (1.5 the equivalent)/THF that (obtains compound (X)) among (-)-DIPCl.Make reaction mixture slowly reach room temperature and spend the night.Steam solvent, then add Et 2O and diethanolamine (2.2 equivalent).Mixture is stirred, finish (by LC-MS, HPLC or TLC monitoring) up to reaction.Leach the precipitation of formation, use Et 2The O washing is then with the filtrate vacuum concentration.In case of necessity, use prepares property HPLC or through flash column chromatography crude product is carried out purifying.
General method E2
Assorted penta ring ((R)-(+)-2-methyl-CBS-oxazaborolidine, the toluene solution of 1M, 0.1-1 equivalent) of (R)-(+)-2-methyl-CBS-oxa-azepine boron is dissolved among the THF, is cooled to 0 ℃ then.Drip borine dimethyl sulphide (Borane-methyl sulfide complex, the THF solution of 2M, 1 equivalent), then reaction mixture is stirred 1h.Reaction mixture is cooled to-10 ℃, lasts 0.5h then and drip the compound (VIII) (1 equivalent) that is dissolved among the THF.The mixture that forms is stirred 1h or up to the reaction end, then temperature slowly risen to 10 ℃.Adding the 1M HCl aqueous solution reacts with cancellation.Add saturated NaHCO 3The aqueous solution is about 8 up to pH.Product is extracted with DCM.The organic extract that merges is through Na 2SO 4Drying, vacuum concentration obtains compound (X) then.Product is optional to carry out purifying through column chromatography.
General method F1
Figure G2007800360541D00191
At 0 ℃ with in argon gas atmosphere, triphenylphosphine (1.3 equivalent)/THF is added among NCS (1.3 equivalent)/THF.The mixture that forms was stirred 30 minutes in envrionment temperature.Add compound (IX) or compound (XI) (1 equivalent) at 0 ℃, reaction mixture is stirred up to reaction in envrionment temperature finish (by LC-MS, HPLC or TLC monitoring) then.Steam solvent, then add hexane, remove precipitation then after filtration.With the filtrate vacuum concentration, in case of necessity, use preparation property HPLC or crude product is carried out purifying through flash column chromatography.
General method F2
Cyanuryl chloride (Cyanuric chloride, 0.6 equivalent) is dissolved in the ethyl acetate.Add DMF (1.5 equivalent), then with mixture stirring at room 10 minutes.Reaction mixture is cooled to 0 ℃.Compound (IX) or compound (X) (1 equivalent) are dissolved in the ethyl acetate, in 10 minutes, are added drop-wise in the reaction mixture then.With the mixture that forms in stirred overnight at room temperature.Add Virahol (about 0.25mL/mmol compound (IX) or compound (X)).Leach precipitation, wash with EtOAc then.Filtrate is concentrated, obtain title compound compound (XI) or compound (XII).
General method G
Sodium borohydride (1 to 2 equivalent) is added among compound (II) (1.0 equivalent)/DMSO.In case stop bubbling, add compound (III) (2-2.5 equivalent).The reaction mixture that forms is finished (by LC-MS, HPLC or TLC monitoring) 40 ℃ of stirrings up to reaction.In case of necessity, use prepares property HPLC or carries out purifying through flash column chromatography.
Embodiment 1
5-{[(1S)-and 1-(5-chloropyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00202
A) 1-(5-chloropyridine-2-yl) ethyl ketone
Figure G2007800360541D00203
(10.7g 77mmol) is dissolved among ether (65mL) and the THF (35mL) with 5-chloropyridine-2-formonitrile HCN in nitrogen atmosphere.With the mixture cooling, it is-63 ℃ up to internal temperature.Last 30 minutes and drip methyl-magnesium-bromide (the THF solution of 3M, 35mL, 105mmol).Then reaction mixture was stirred 45 minutes at-60 ℃, be warmed to room temperature then.Add 50mL THF to dissolve any sedimentary material.Behind room temperature reaction 1h, judge that through HPLC reaction finishes.(aqueous solution 100mL), stirs 4h with reaction mixture then to add 2M hydrochloric acid.Then with sodium bicarbonate with pH regulator to 7.Separate each phase, obtain product with DCM from the aqueous phase extracting twice then.The organic extract that merges is through dried over sodium sulfate, then vacuum concentration.(eluent: heptane: the ethyl acetate gradient) purifying obtains 7.9g (64% yield) title compound to product through flash column chromatography.
1H?NMR(300MHz，CDCl 3)：δppm?8.62(m，1H)；8.00(m，1H)；7.80(m，1H)；2.70(s，3H)。
B) (1S)-1-(5-chloropyridine-2-yl) ethanol
Figure G2007800360541D00211
Title compound is by general method E2, and (780mg 5mmol) begins to prepare from 1-(5-chloropyridine-2-yl) ethyl ketone.Through the flash column chromatography purifying, obtain 695mg (88% yield) title compound, 92%ee.
1H NMR (300MHz, CDCl 3): δ ppm 8.47 (s, 1H); 7.65 (d, 1H); 7.26 (d, 1H); 4.87 (q, 1H); 3.87 (br s, 1H); 1.47 (d, 3H); MS (ESI) m/z 140 and 142[M+1] +
C) 5-chloro-2-[(1R)-and the 1-chloroethyl] pyridine
Figure G2007800360541D00212
Title compound is by general method F2, and (695mg 4.41mmol) begins to prepare from (1S)-1-(5-chloropyridine-2-yl) ethanol.Crude product is not purified just to be used for next step.
1H NMR (400MHz, CDCl 3): δ ppm 8.46 (d, 1H), 7.64 (dd, 1H), 7.41 (d, 1H), 5.08 (q, 1H), 1.80 (d, 3H); MS (ESI) m/z 176 and 178[M+1] +
D) (2R)-and 2-[(2-amino-5-{[(1S)-1-(5-chloropyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00213
Title compound is by general method A, from (2R)-2-[(2-amino-5-sulfydryl [1,3] amino thiazole [4,5-d] pyrimidin-7-yl also)]-4-methylpent-1-alcohol (823mg, 2.75mmol) and 5-chloro-2-[(1R)-1-chloroethyl] pyridine (＜4.4mmol) begin to prepare.(eluent: DCM: the methyl alcohol gradient) purifying obtains 350mg (30% yield) title compound through flash column chromatography.
1H NMR (400MHz, CD 3OD): δ ppm 8.49 (d, 1H), 7.79 (dd, 1H), 7.66 (d, 1H), 5.22 (q, 1H), 4.46 (br s, 1H), 3.40-3.57 (m, 2H), 1.66-1.78 (m, 4H), 1.40-1.61 (m, 2H), 0.93-1.03 (m, 6H); MS (ESI) m/z 439 and 441[M+1] +
E) (2R)-2-[(2-chloro-5-{[(1S)-1-(5-chloropyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00221
Title compound is by general method B, from (2R)-2-[(2-amino-5-{[(1S)-1-(5-chloropyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-(340mg 0.77mmol) begins to prepare 4-methylpent-1-alcohol.
MS (ESI) m/z 458 and 460[M+1] +
F) (2R)-2-[(5-{[(1S)-and 1-(5-chloropyridine-2-yl) ethyl] sulfenyl }-2-methoxyl group [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00222
Title compound is to use general method C, (the 2R)-2-[(2-chloro-5-{[(1S that obtains from previous step)-and 1-(5-chloropyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, except reaction mixture being heated to 50 ℃ and lasting 1h.After reaction finished, with the reaction mixture dilute with water, product extracted (four times) with DCM.The organic extract that merges is through dried over sodium sulfate, and vacuum concentration obtains title compound then, and it is not purified just to be used for next step.
MS (ESI) m/z 453 and 455[M+1] +
G) 5-{[(1S)-and 1-(5-chloropyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00231
Title compound is to use general method D, (2R)-2-[(5-{[(1S)-1-(5-chloropyridine-2-yl) ethyl that obtains from previous step] sulfenyl }-2-methoxyl group [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is stirred 2.5h at 50 ℃, then in stirred overnight at room temperature.Reaction is diluted reaction mixture after finishing with salt solution, then with DCM extraction (three times).The organic extract that merges is through dried over sodium sulfate and vacuum concentration.(eluent DCM: the methyl alcohol gradient) purifying obtains 160mg to product through flash chromatography.Through preparation property HPLC (post: Chiralcel OJ; Eluent: ethanol/heptane 30/70; Flow velocity: 12ml/min) be further purified, obtain the 82mg title compound.
1H NMR (400MHz, CD 3OD): δ ppm 8.24 (d, 1H), 7.56 (dd, 1H), 7.38 (d, 1H), 4.90 (q, 1H), 4.19 (br s, 1H), 3.16-3.30 (m, 2H), 1.39-1.51 (m, 4H), 1.15-1.34 (m, 2H), 0.68-0.76 (m, 6H); 1H NMR (DMSO-d 6) δ ppm 12.36 (br s, 1H), 8.57 (d, 1H), 7.86 (dd, 1H); 7.57 (d, 1H); 7.23 (d, 1H); 5.03 (q, 1H); 4.69 (t, 1H); 4.29 (br s, 1H); 3.40-3.25 (m, 2H), 1.66 (d, 3H), 1.63-1.52 (m, 1H); 1.48-1.32 (m, 2H), 0.88 (d, 3H), 0.85 (d, 3H); MS (ESI) m/z 440 and 442[M+1] +, 438 and 440[M-1] +
Embodiment 2
5-{[(1S)-and 1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00232
A) 1-(5-fluorine pyridine-2-yl) ethyl ketone
Figure G2007800360541D00241
In nitrogen atmosphere, (29g 240mmol) is dissolved among the THF (150mL) with 5-fluoro-pyridine-2-formonitrile HCN.Reaction mixture is cooled to internal temperature is-64 ℃.Last 40 minutes and drip methyl-magnesium-bromide (the THF solution of 3M, 105mL, 315mmol).Reaction mixture is stirred 1.5h at-65 ℃, be warmed to room temperature then.Add THF (50mL), then with mixture restir 3h.Add 2M hydrochloric acid (aqueous solution 100mL), is subacidity up to mixture, then with reaction mixture in stirred overnight at room temperature.Add sodium bicarbonate then with the neutralization reaction mixture.Separate each phase, water extracts with DCM.The organic extract salt water washing that merges is through dried over sodium sulfate and vacuum concentration.Crude product obtains 18g (55% yield) title compound through the flash column chromatography purifying.
1H?NMR(300MHz，CDCl 3)：δppm?8.50(m，1H)；8.10(m，1H)；7.52(m，1H)；2.70(s，3H)。
B) (1S)-1-(5-fluorine pyridine-2-yl) ethanol
Figure G2007800360541D00242
Title compound is by general method E2, and (3.18g 22.9mmol) begins to prepare from 1-(5-fluorine pyridine-2-yl) ethyl ketone.Through the flash column chromatography purifying, obtain 2.73g (84% yield) title compound, 84%ee.
1H?NMR(300MHz，CDCl 3)：δppm?8.38(m，1H)；7.5-7.2(m，2H)；4.89(q，1H)；3.9(br?s，1H)；1.49(d，3H)。
C) 2-[(1R)-the 1-chloroethyl]-5-fluorine pyridine
Figure G2007800360541D00243
Title compound with 80%ee is by general method F2, and (720mg 5.1mmol) begins to prepare from (1S)-1-(5-fluorine pyridine-2-yl) ethanol.Crude product is not purified just to be used for next step.
1H?NMR(300MHz，CDCl 3)：δppm?8.44-8.40(m，1H)；7.6-7.4(m，2H)；5.16(q，1H)，1.86(d，3H)。
D) (2R)-and 2-[(2-amino-5-{[(1S)-1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00251
Title compound is by general method A, from (2R)-2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol (940mg, 3.1mmol) and 2-[(1R)-the 1-chloroethyl]-(0.81g 5.1mmol) begins to prepare 5-fluorine pyridine.Product obtains 0.75g (56% yield) title compound through the flash column chromatography purifying.
1H?NMR(400MHz，DMSO-d 6)：δppm?8.51(d，1H)，7.98(s，2H)，7.65(dt，1H)；7.58(dd，1H)，6.88(d，1H)；5.12(q，1H)；4.66(t，1H)；4.27(br?s，1H)；3.41-3.27(m，2H)，1.66(d，3H)，1.65-1.55(m，1H)；1.48-1.35(m，2H)，0.88(d，3H)，0.85(d，3H)；MS(ESI)m/z?423[M+1] +。
E) (2R)-2-[(2-chloro-5-{[(1S)-1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00252
Title compound is to use general method B, from (2R)-2-[(2-amino-5-{[(1S)-1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] amino thiazole [4,5-d] pyrimidin-7-yl also)]-(750mg 1.77mmol) begins to prepare 4-methylpent-1-alcohol.
MS (ESI) m/z 442 and 444[M+1] +
F) (2R)-2-[(5-{[(1S)-and 1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl }-2-methoxyl group [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00261
Title compound is to use general method C, (the 2R)-2-[(2-chloro-5-{[(1S that obtains from previous step)-and 1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is heated to 50 ℃ and lasting 1.5h.After reaction finishes, with reaction mixture water and salt solution dilution, product chloroform extraction (three times).The organic extract liquid that merges is through dried over mgso, and vacuum concentration obtains title compound then, its not purified just use.
MS(ESI)m/z?438[M+1] +。
G) 5-{[(1S)-and 1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00262
Title compound is to use general method D, (2R)-2-[(5-{[(1S)-1-(the 5-fluorine pyridine-2-yl) ethyl that obtains from previous step] sulfenyl }-2-methoxyl group [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is stirred 3h at 50 ℃.Reaction is diluted reaction mixture after finishing with salt solution, then with DCM extraction (three times).The organic extract that merges is through dried over mgso and vacuum concentration.Product is through purification by flash chromatography (eluent: DCM: the methyl alcohol gradient).Through preparation property HPLC (post Chiralcel OJ; Eluent: ethanol; Flow velocity: 8mL/min) be further purified, obtain the 13mg title compound.
1H?NMR(CD 3OD)：δppm?8.19(d，1H)，7.46(dd，1H)，7.36(dt，1H)，4.97(q，1H)，4.26(br?s，1H)，3.23-3.34(m，2H)，1.44-1.55(m，4H)，1.19-1.37(m，2H)，0.75(dd，6H)； 1H?NMR(DMSO-d 6)δppm?12.36(br?s，1H)，8.52(d，1H)，7.66(dt，1H)；7.60(dd，1H)，7.23(d，1H)；5.07(q，1H)；4.69(t，1H)；4.30(br?s，1H)；?3.40-3.26(m，2H)，1.67(d，3H)，1.64-1.53(m，1H)；1.48-1.33(m，2H)，0.88(d，3H)，0.85(d，3H)；MS(ESI)m/z?424[M+1] +。
Embodiment 3
5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00271
A) 4-(1-chloroethyl)-3-fluorine pyridine
(0.8g 5.7mmol) handles with thionyl chloride (5mL), then with mixture heating up to the 80 ℃ also lasting 2h that forms with 1-(3-fluorine pyridin-4-yl) ethanol.Add entry (10mL) and saturated sodium bicarbonate (aqueous solution, 10mL).Product is extracted (three times) with DCM.The organic extract salt water washing that merges is through dried over sodium sulfate and vacuum concentration.(eluent: heptane: the ethyl acetate gradient) purifying obtains 0.36g (39% yield) title compound to crude product through flash column chromatography.
1H?NMR(300MHz，CDCl 3)：δppm?8.45(m，2H)，7.50(m，1H)，5.34(q，1H)，1.83(d，3H)。
B) (2R)-and 2-[(2-amino-5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00273
Title compound (370mg, 47% yield) is to use general method A, from (2R)-2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-(560mg 1.87mmol) begins to prepare 4-methylpent-1-alcohol.
MS(ESI)m/z?423[M+1] +。
C) (2R)-and 2-[(2-chloro-5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00281
Title compound is to use general method B, from (2R)-2-[(2-amino-5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-(370mg 0.84mmol) begins to prepare 4-methylpent-1-alcohol.
D) (2R)-and 2-[(5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-2-methoxyl group [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00282
Title compound is to use general method C, (2R)-2-[(2-chloro-5-{[1-(the 3-fluorine pyridin-4-yl) ethyl that obtains from previous step] sulfenyl } [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is heated to 50 ℃ and lasting 1.5h.After reaction finishes, reaction mixture water and salt solution (1: 1) dilution, product extracts (twice) with DCM.With the pH regulator to 7 of ammonium chloride with water, product extracts (twice) with DCM then.The organic extract that merges obtains title compound through dried over sodium sulfate and vacuum concentration.
E) 5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Title compound is to use general method D, (2R)-2-[(5-{[1-(the 3-fluorine pyridin-4-yl) ethyl that obtains from previous step] sulfenyl }-2-methoxyl group [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is stirred 2h at 50 ℃.After reaction finishes, reaction mixture with saturated sodium bicarbonate aqueous solution and water (1: 1) dilution, is extracted (three times) with DCM then.The organic extract that merges is through dried over sodium sulfate and vacuum concentration.(eluent: heptane: the ethyl acetate gradient), obtain title compound, it is the mixture (194mg) of diastereomer to product through the flash column chromatography purifying.
MS(ESI)m/z424[M+1] +。
Embodiment 4
5-{[(1S)-and 1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00291
A) 1-(6-bromo-3-fluoro-pyridine-2-yl) ethyl ketone
Figure G2007800360541D00292
(11g 62.5mmol) is dissolved in the ether with 2-bromo-5-fluoro-pyridine in room temperature with in nitrogen atmosphere.Is-66 ℃ with the reaction mixture cooling up to internal temperature.Last 0.5h drip butyllithium (hexane solution of 2.5M, 26mL, 65mmol).The reaction mixture that forms is stirred 1h at-65 ℃.(6.5mL 70mmol), stirs 2h with reaction mixture at-65 ℃ then to last 10 minutes adding N,N-dimethylacetamide.Add 1M aqueous hydrochloric acid (50mL), then mixture is warmed to room temperature.With extra hydrochloric acid with pH regulator to 7.Water extracted with diethyl ether 3 times.The organic phase salt water washing that merges, through dried over sodium sulfate, and vacuum concentration.(eluent: heptane: the ether gradient) purifying obtains 4.6g (34% yield) title compound through flash column chromatography.
1H NMR (300MHz, DMSO-d 6): δ ppm 8.0-7.8 (m, 2H); 2.57 (s, 3H); MS (ESI) m/z 218 and 220[M+1] +
B) (1S)-1-(6-bromo-3-fluoro-pyridine-2-yl) ethanol
Figure G2007800360541D00293
Title compound is to use general method E2, and (1.76g 8.19mmol) begins to prepare from 1-(6-bromo-3-fluoro-pyridine-2-yl) ethyl ketone.(eluent: heptane: the ethyl acetate gradient) purifying obtains 1.31g (73% yield) title compound, 80%ee to product through flash column chromatography.
1H NMR (300MHz, CDCl 3): δ ppm 7.38 (m, 1H); 7.26 (m, 1H); 5.06 (q, 1H); 3.38 (br s, 1H); 1.47 (d, 3H); MS (ESI) m/z 220 and 222[M+1] +, m/z 202[M-H2O] +
C) (1S)-1-(3-fluoro-pyridine-2-yl) ethanol
Figure G2007800360541D00301
With (1S)-1-(6-bromo-3-fluoro-pyridine-2-yl) ethanol (1.3g, 5.9mmol), triethylamine (1.6mL, 11.5mmol) and palladium/carbon (0.64g 0.34mmol) mixes in DCM (25mL).Flask is vacuumized/charge into hydrogen, repeat this operation 4 times, place 24h under the 2.5 normal atmosphere hydrogen in room temperature then.Mixture is filtered, and solid washs with DCM.Filtrate water and salt water washing are through dried over sodium sulfate and vacuum concentration.(eluent: DCM: the methyl alcohol gradient) purifying obtains 0.54g (65% yield) title compound to crude product through flash column chromatography.
1H?NMR(300MHz，CDCl 3)：δppm?8.38(m，1H)；7.39(m，1H)；7.26(m，1H)；5.11(q，1H)；4.16(br?s，1H)；1.49(d，3H)。
D) 2-((R)-1-chloroethyl)-3-fluoro-pyridine
Figure G2007800360541D00302
Title compound (0.24g) is to use general method F2, and (254mg 1.8mmol) begins to prepare from (1S)-1-(3-fluoro-pyridine-2-yl) ethanol.
1H NMR (300MHz, CDCl 3): δ ppm 8.46 (m, 1H); 7.47 (m, 1H); 7.34 (m, 1H); 5.48 (q, 1H), 1.94 (d, 3H); MS (ESI) m/z160 and 162[M+1] +
E) (2R)-and 2-[(2-amino-5-{[(1S)-1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00303
Title compound is to use general method A, from (2R)-2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol (348mg, 1.16mmol) and 2-((R)-1-chloroethyl)-3-fluoro-pyridine (240mg 1.5mmol) begins to prepare.(eluent: DCM: the methyl alcohol gradient) purifying obtains 190mg (47% yield) title compound, diastereomeric excess 60% through flash column chromatography.
1H?NMR(400MHz，DMSO-d 6)：δppm?8.40(dt，1H)，7.98(s，2H)，7.70(m，1H)，7.40(m，1H)；6.92(d，1H)；5.45(q，1H)；4.65(t，1H)；4.27(br?s，1H)；3.45-3.30(m，2H)，1.69(d，3H)，1.66-1.58(m，1H)，1.50-1.35(m，2H)，0.88(d，3H)，0.85(d，3H)；MS(ESI)m/z?423[M+1] +。MS(ESI)m/z?423[M+1] +。
F) (2R)-2-[(2-chloro-5-{[(1S)-1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00311
Title compound is to use general method B, from (2R)-2-[(2-amino-5-{[(1S)-1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] amino thiazole [4,5-d] pyrimidin-7-yl also)]-(135mg 0.32mmol) begins to prepare 4-methylpent-1-alcohol.
MS (ESI) m/z 442 and 444[M+1] +
G) (2R)-2-[(5-{[(1S)-and 1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl }-2-methoxyl group [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino]-4-methylpent-1-alcohol
Figure G2007800360541D00312
Title compound is to use general method C, (the 2R)-2-[(2-chloro-5-{[(1S that obtains from previous step)-and 1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl } [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is heated to 50 ℃ and lasting 1.5h.After reaction finishes, with reaction mixture water and salt solution (2: 1) dilution, product chloroform extraction (three times).The organic extract that merges obtains title compound through dried over mgso and vacuum concentration.
MS(ESI)m/z?438[M+1] +。
H) 5-{[(1S)-and 1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00321
Title compound is to use general method D, from (2R)-2-[(5-{[(1S)-1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl }-2-methoxyl group [1,3] thiazole also [4,5-d] pyrimidin-7-yl) amino]-4-methylpent-1-alcohol preparation, different is that reaction mixture is heated to 50 ℃ and lasting 1.5h.Reaction is diluted reaction mixture after finishing with salt solution, then with DCM extraction (three times).The organic extract that merges is through dried over mgso and vacuum concentration.The methyl alcohol gradient) and preparation property HPLC purifying product is (eluent: DCM:, obtain the 20mg title compound earlier after flash column chromatography.
1H?NMR(DMSO-d 6)：δppm?12.37(br?s，1H)，8.41(dt，1H)，7.72(m，1H)；7.42(m，1H)；7.27(br?s，1H)；5.43(q，1H)；4.67(t，1H)；4.30(br?s，1H)；3.44-3.30(m，2H)，1.70(d，3H)，1.65-1.55(m，1H)；1.52-1.32(m，2H)，0.89(d，3H)，0.86(d，3H)；MS(ESI)m/z?424[M+1] +。
Embodiment 5
2-{[(1S)-1-[(7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino }-2-oxo-2,3-dihydro [1,3] thiazole is [4,5-d] pyrimidine-5-yl also) sulfenyl] ethyl } pyridine-4-formonitrile HCN
Figure G2007800360541D00322
A) 2-((S)-1-hydroxyl-ethyl)-pyridine-4-formonitrile HCN
Figure G2007800360541D00331
Title compound is according to general method E1, from 2-ethanoyl-pyridine-4-formonitrile HCN (1.42g, 9.72mmol) and (-)-DIPCl (4.67g 14.57mmol) begins to prepare.
1H?NMR(500MHz，CDCl 3)：δppm?8.72(d，1H)，7.62(s，1H)，7.44(dd，1H)，4.96(q，1H)，1.54(d，3H)。
B) 2-((R)-1-chloro-ethyl)-pyridine-4-formonitrile HCN
Figure G2007800360541D00332
(32.2mg is according to general method F1 0.19mmol) to title compound, and (400mg 2.7mmol) begins to prepare from 2-((S)-1-hydroxyl-ethyl)-pyridine-4-formonitrile HCN.
1H?NMR(500MHz，CDCl 3)：δppm?8.74(d，1H)，7.76(s，1H)，7.46(dd，1H)，5.16(q，1H)，1.88(d，3H)。
C) (2R)-2-{2-chloro-5-[2-chloro-7-((1R)-1-methylol-3-methyl-butyl amino)-thiazole [4,5-d] pyrimidine-5-base dithio-also]-thiazole [4,5-d] pyrimidin-7-yl amino also }-4-methyl-penta-1-alcohol
Figure G2007800360541D00333
At 0 ℃, with Sodium Nitrite (5.19g, 75mmol) drips of solution in water (25mL) is added to (2R)-2-[[2-amino-5-sulfydryl [1,3] thiazole also [4,5-d] pyrimidin-7-yl] amino]-(7.50g is 25mmol) in the solution in concentrated hydrochloric acid (150mL) and acetonitrile (150mL) for 4-methylpent-1-alcohol.Reaction mixture is stirred 18h at 0-5 ℃, be poured on then on the ice (500mL), use ethyl acetate extraction.Leach any remaining solid.The organic phase that merges is successively with salt solution and saturated sodium bicarbonate aqueous solution washing.Dry and the evaporation with organic phase is then to wherein adding the solid that had before leached.With all solids pulp in ethyl acetate, filter afterwards and obtain title compound (6.3g, 80% yield).
1H?NMR(DMSO-d 6)：δppm?8.25(d，2H)，4.19(m，2H)，3.35(m，4H)，1.40(m，4H)，1.21(m，2H)，0.68(d，6H)，0.51(d，6H)；MS(ESI)m/z?635[M+1] +。
D) (2R)-2-{5-[7-((1R)-1-methylol-3-methyl-butyl amino)-2-methoxyl group-thiazole [4,5-d] pyrimidine-5-base dithio-also]-2-methoxyl group-thiazole [4,5-d] pyrimidin-7-yl amino also }-4-methyl-penta-1-alcohol
Figure G2007800360541D00341
At 0 ℃, with potassium hydroxide (0.53g, 9.4mmol) solution in methyl alcohol (5mL) is added to (2R)-2-{2-chloro-5-[2-chloro-7-((1R)-1-methylol-3-methyl-butyl amino)-thiazole also [4,5-d] pyrimidine-5-base dithio-]-thiazole also [4,5-d] pyrimidin-7-yl amino }-(3.0g is 4.7mmol) in the solution in methyl alcohol (200mL) for 4-methyl-penta-1-alcohol.Reaction mixture is kept 18h at 0-5 ℃.Steam solvent, resistates absorbs with methanol/ethyl acetate (1: 1).This solution is carried out chromatogram fast, and (eluent: ethyl acetate) purifying obtains title compound (2.0g, 68% yield).
MS(ESI)m/z?627[M+1] +。
E) 5-[7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino }-[1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone-5-base dithio-also]-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00342
The mixture of concentrated hydrochloric acid (20mL) and water (20mL) is added to (2R)-2-{5-[7-((1R)-1-methylol-3-methyl-butyl amino)-2-methoxyl group-thiazole also [4,5-d] pyrimidine-5-base dithio-]-2-methoxyl group-thiazole also [4,5-d] pyrimidin-7-yl amino }-4-methyl-penta-1-alcohol (1.5g, 2.4mmol) in 1, in the solution in the 4-dioxane (20mL).Then solution is stirred 18h at 45 ℃.Steam solvent, the resistates up in ethyl acetate.Collect any insoluble resistates after filtration.Make filtrate carry out flash column chromatography (eluent: ethyl acetate: methyl alcohol 95: 5).To pool together from solid residue and the product that chromatography is collected, obtain title compound (600mg, 42% yield).
1H?NMR(DMSO-d 6)：δppm?12.45(s，2H)，7.33(d，2H)，4.62(t，2H)，4.17(brs，2H)，1.48-1.31(m，4H)，1.25-1.14(m，2H)，0.72(d，6H)，0.56(d，6H)；MS(ESI)m/z?599[M+1] +。
F) 2-{ (1S)-1-[(7-{[(1R)-1-(methylol)-3-methyl butyl] amino }-2-oxo-2,3-dihydro [1,3] thiazole is [4,5-d] pyrimidine-5-yl also) sulfenyl] ethyl } pyridine-4-formonitrile HCN
Figure G2007800360541D00351
Title compound is according to general method G, and adding DIPEA (2 equivalent) preparation.From 5,5 '-dithio-two [7-{[(1R)-1-(methylol)-3-methyl butyl] amino } [1,3] thiazole also [4,5-d] pyrimidine-2 (3H)-ketone] (64mg, 0.096mmol) and 2-((R)-1-chloro-ethyl)-pyridine-4-formonitrile HCN (32mg, 0.192mmol) beginning, obtaining title compound (39mg), it is a non-enantiomer mixture.(post: Kromasil-C18) purifying obtains 15mg (36% yield) title compound, 98%de through preparation property HPLC.
1H?NMR(500MHz，CD 3OD)：δppm?8.71(d，1H)，7.92(s，1H)，7.56(d，1H)，5.17(q，1H)，4.4(s，1H)，3.40-3.52(m，2H)，1.72(d?3H)，1.60-1.71(m?1H)，1.38-1.54(m，2H)，0.90-0.98(m?6H)；MS(ESI +)m/z?431[M+H] +。
Embodiment 6
5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol) butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00352
A) (2R)-2-{[2-amino-5-(benzylthio-) [1,3] thiazole [4,5-d] pyrimidin-7-yl also] amino } penta-1-alcohol
Figure G2007800360541D00353
With 5-(benzylthio-)-7-chlorine [1,3] thiazole also [4,5-d] pyrimidine-2-amine (6.0g 19.4mmol) is dissolved among the NMP (30mL).Add DIPEA (8.4mL, 48.5mmol) and 2-amino-(2R)-the 1-amylalcohol (3.5g, 33.9mmol), then with mixture heating up to 110 ℃ and continue 4 days.After being cooled to room temperature, mixture is poured in the water (200mL).Filter the product that collecting precipitation goes out, wash with water, just be used for next step (7.0g, 97% yield) without being further purified.
MS(ESI +)m/z?376[M+H] +。
B) (2R)-and 2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino] penta-1-alcohol
Figure G2007800360541D00361
Round-bottomed flask is equipped with dry ice-ethanol condenser, immerses then in dry ice-ethanol cooling bath.Ammonia (250mL) condensation is entered in the flask, then adds also [4,5-d] pyrimidin-7-yl of (2R)-2-{[2-amino-5-(benzyl sulfenyl) [1,3] thiazole] amino } penta-1-alcohol (6.8g, 18.1mmol).The mixture of formation is warmed to-33 ℃, the sodium Metal 99.5 that adds the fritter form then is up to occurring blue and continuing 30 seconds.To react cancellation by adding spoonful solid ammonium chloride then.Steam ammonia, then water (250mL) is added in the resistates.The mixture of gained is neutralized with 1M hydrochloric acid (aqueous solution).Filter the product that collecting precipitation goes out, wash with water, vacuum-drying then obtains 4.15g (80% yield) title compound.
MS(ESI +)m/z?286[M+H] +。
C) (2R)-2-{2-chloro-5-[2-chloro-7-((1R)-1-methylol-butyl amino)-thiazole [4,5-d] pyrimidine-5-base dithio-also]-thiazole [4,5-d] pyrimidin-7-yl amino also }-penta-1-alcohol
With (2R)-2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) amino] (4.0g 14mmol) is dissolved in acetonitrile (100ml) and the concentrated hydrochloric acid (150mL) penta-1-alcohol.(1.93g, 28mmol) (10mL) soluble in water is then 0 ℃ of adding with Sodium Nitrite.Place 0 ℃ to continue 2 days reaction mixture up to finishing through the LCMS monitoring reaction.Reaction mixture is poured on ice, filters the product that collecting precipitation goes out then.Solid vacuum-drying is obtained 3.3g (78% yield) title compound.
1H?NMR(DMSO-d 6)：δppm?8.27(d，1H)，4.32-3.81(m，2H)，3.50-3.23(m，2H)，1.37-1.19(m，2H)，1.10-0.93(m，1H)，0.94-0.78(m，1H)，0.49(t，3H)；?MS(ESI)m/z?607[M+1] +。
D) (2R)-2-{5-[7-((1R)-1-methylol-butyl amino)-2-methoxyl group-thiazole [4,5-d] pyrimidine-5-base dithio-also]-2-methoxyl group-thiazole [4,5-d] pyrimidin-7-yl amino also }-penta-1-alcohol
Figure G2007800360541D00371
At 0 ℃, with potassium hydroxide (495mg, 8.8mmol) be added to (2R)-2-{2-chloro-5-[2-chloro-7-((1R)-1-methylol-butyl amino)-thiazole also [4,5-d] pyrimidine-5-base dithio-]-thiazole also [4,5-d] pyrimidin-7-yl amino }-(2.68g is 4.41mmol) in the solution in methyl alcohol (200mL) for penta-1-alcohol.Reaction mixture is spent the night 0 ℃ of stirring, steam methyl alcohol then.Resistates is poured in the water, filtered then and collect the precipitation that forms.Moist crude product just is used for next step without any being further purified.
MS(ESI)m/z?599[M+1] +。
E) 5-[7-{[(1R)-and 1-(methylol)] amino }-[1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone-5-base dithio-also]-7-{[(1R)-and 1-(methylol-butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00372
Rough (2R)-2-{5-[7-((1R)-1-methylol-butyl amino)-2-methoxyl group-thiazole that will obtain from previous step also [4,5-d] pyrimidine-5-base dithio-]-2-methoxyl group-thiazole also [4,5-d] pyrimidin-7-yl amino }-penta-1-alcohol (4.41mmol) is dissolved in 1, in the 4-dioxane (100mL).Add concentrated hydrochloric acid (2mL) and water (2mL), then the mixture that forms is spent the night 45 ℃ of stirrings.Vacuum evaporating solvent is settled out product by adding entry.Filter collecting precipitation, wash with water then.(eluent: DCM: the ethyl acetate gradient) purifying obtains 1.5g (two step yields are 59%) title compound to crude product through flash column chromatography.
1H?NMR(DMSO-d 6)：δppm?12.46(s，1H)，7.33(d，1H)，4.61(t，1H)，4.10(br.s.，1H)，3.35(t，2H)，1.37-1.20(m，2H)，1.13-1.10(m，1H)，0.96-0.82(m，1H)，0.59(t，3H)；MS(ESI)m/z?571[M+1] +。
F) (1S)-1-(6-chloropyridine-3-yl) ethanol
Figure G2007800360541D00373
Title compound is according to general method E1, and (0.80g 5.14mmol) prepares, and obtains 0.71g (88% yield) title compound to use 1-(6-chloropyridine-3-yl) ethyl ketone.
1H NMR (CDCl 3): δ ppm 8.40-8.28 (m, 1H), 7.75-7.63 (m, 1H), 7.35-7.24 (m, 1H), 5.04-4.79 (m, 1H), 1.63-1.45 (m, 3H); MS (ESI) m/z158 and 160[M+1] +
G) 2-chloro-5-[(1R)-and the 1-chloroethyl] pyridine
Figure G2007800360541D00381
Title compound is according to general method F1, and (0.20g 1.27mmol) prepares, and obtains 0.16g (72% yield) title compound to use (1S)-1-(6-chloropyridine-3-yl) ethanol.
1H NMR (CDCl 3): δ ppm 8.45-8.35 (m, 1H), 7.79-7.70 (m, 1H), 7.39-7.29 (m, 1H), 5.07 (q, 1H), 1.85-1.78 (m, 3H); MS (ESI) m/z 176 and 178[M+1] +
H) 5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol) butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00382
Title compound is according to general method G, use 5-[7-{[(1R)-1-(methylol)] amino }-[1,3] thiazole also [4,5-d] pyrimidine-2 (3H)-ketone-5-base dithio-]-7-{[(1R)-and 1-(methylol-butyl] amino } [1,3] thiazole also [4,5-d] pyrimidine-2 (3H)-ketone (0.10g, 0.175mmol), 2-chloro-5-[(1R)-the 1-chloroethyl] pyridine (0.069g, 0.39mmol) and sodium borohydride (0.040g, 1.05mmol) preparation, obtain 0.055g (37% yield) title compound.
1H?NMR(CDCl 3)：δppm?8.52-8.38(m，1H)，7.87-7.72(m，1H)，7.30-7.26(m，1H)，4.91-4.81(m，1H)，4.74-4.65(m，1H)，4.29-4.17(m，1H)，3.68-3.52(m，2H)，1.69-1.64(m，3H)，1.56-1.46(m，2H)，1.46-1.32(m，2H)，0.98-0.90(m，3H)；
MS (ESI) m/z 426 and 428[M+1] +
Embodiment 7
5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-[[(1R)-and 1-(methylol) butyl] (methyl) amino] [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00391
A) N-(ethoxy carbonyl)-D-norvaline
Figure G2007800360541D00392
With the D-norvaline (10.0g, 85.3mmol) be dissolved in aqueous sodium hydroxide solution (4M, 25mL) in.Last 15 minutes 0 ℃ add Vinyl chloroformate (10.6mL, 111mmol) and aqueous sodium hydroxide solution (4M, 25mL).Reaction mixture is warmed to room temperature, stirs 4h in this temperature then.Reaction mixture with ether washing three times, is used aqueous hydrochloric acid (2M) acidifying then.Product extracted with diethyl ether three times.The organic phase that merges obtains title compound through dried over mgso and vacuum concentration with quantitative yield.
1H NMR (CDCl 3): δ ppm 6.43 (br s, 1H), 5.22 (d, 1H), 4.37 (q, 1H), 4.13 (q, 2H), 1.84 (m, 1H), 1.68 (sextet, 1H), 1.42 (sextet, 1H), 1.25 (t, 3H), 0.95 (t, 3H); MS (CI) 144 (100%), 190[M+1] +
B) (2R)-2-(methylamino) penta-1-alcohol
Figure G2007800360541D00393
0 ℃ in nitrogen atmosphere, (6.5g 171mmol) is suspended among the THF with lithium aluminum hydride.N-(ethoxy carbonyl)-D-norvaline is dissolved among the THF, then 0 ℃ of dropping.Reaction mixture refluxed is spent the night.After being cooled to room temperature, add saturated aqueous sodium sulfate to form slurry.The mixture of gained is through diatomite filtration.Solid washs with DCM, up to extracting all products.The filtrate that merges is through dried over sodium sulfate and vacuum concentration.Steep-steep distillation (Bulb-to-bulbdestillation) at 0.1 millibar, the cut between collecting 75-85 ℃ obtains 7.1g (71% yield) title compound.
1H?NMR(CDCl 3)：δppm?3.63(dd，1H)；3.30(dd，1H)；2.51(m，1H)；2.41(s，3H)；2.09(br?s，2H)；1.50-1.28(m，4H)；0.93(t，3H)；MS(CI)86(100％)，118[M+1] +。
C) (2R)-and 2-{[2-amino-5-(benzyl sulfenyl) [1,3] thiazole [4,5-d] pyrimidin-7-yl also] (methyl) amino } penta-1-alcohol
With 5-(benzyl sulfenyl)-7-chlorine [1,3] thiazole also [4,5-d] pyrimidine-2-amine (6.0g 19.4mmol) is dissolved among the NMP (25mL).Add DIPEA (6.8mL, 38.8mmol) and (2R)-2-(methylamino) penta-1-alcohol (3.4g, 29.1mmol), then with mixture heating up to 120 ℃ and continue 3 days.Add extra (2R)-2-(methylamino) penta-1-alcohol (350mg, 2.99mmol) and DIPEA (1mL, 5.74mmol), then with reaction mixture 120 ℃ the heating 6h.After being cooled to room temperature, mixture is poured in the ice.The product that goes out of collecting precipitation after filtration, (eluent DCM: the ethyl acetate gradient) purifying obtains title compound (5.74g, 76% yield) through flash column chromatography then.
1H?NMR(DMSO-d 6)：δppm?7.98(br?s，2H)，7.41(m，2H)，7.29(m，2H)，7.22(m，1H)，4.73(t，1H)，4.54(br?s，1H)，4.33(m，2H)，3.55-3.40(m，2H)，3.01(s，3II)，1.52-1.44(m，2H)，1.25-1.10(m，2H)，0.84(t，3H)；MS(ESI)m/z?390[M+1] +。
D) (2R)-and 2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) (methyl) amino] penta-1-alcohol
Figure G2007800360541D00402
Round-bottomed flask is equipped with dry ice-ethanol condenser, immerses then in dry ice-ethanol cooling bath.Ammonia (200mL) condensation is entered in the flask, then adds also [4,5-d] pyrimidin-7-yl of (2R)-2-{[2-amino-5-(benzylthio-) [1,3] thiazole] (methyl) amino } penta-1-alcohol (5.43g, 13.9mmol).The mixture of formation is warmed to-33 ℃, the sodium Metal 99.5 that adds the fritter form then is up to occurring blue and continuing 30 seconds.To react cancellation by adding spoonful solid ammonium chloride then.Steam ammonia, then water (250mL) is added in the resistates.The mixture that forms neutralizes with 1M hydrochloric acid (aqueous solution).Filter the product that collecting precipitation goes out, water and acetonitrile washing, vacuum-drying then obtains 3.38g (81% yield) title compound.
1H?NMR(DMSO-d 6)：δppm?12.81(br?s，1H)；8.45(br?s，2H)，4.84(br?s，1H)，?3.55-3.40(m，2H)，3.02(s，3H)，1.48(m，2H)，1.21(m，2H)，0.87(t，3H)；MS(ESI)m/z?300[M+1] +。
E) (2R, 2 ' R)-2,2 '-{ dithio-two [(2-chlorine [1,3] thiazole is [4,5-d] pyrimidine-5 also, 7-two bases) (methylene imine base)] } two (penta-1-alcohol)
With (2R)-2-[(2-amino-5-sulfydryl [1,3] thiazole [4,5-d] pyrimidin-7-yl also) (methyl) amino] (1.0g 3.34mmol) is dissolved in acetonitrile (25ml) and the concentrated hydrochloric acid (40mL) penta-1-alcohol.(461mg 6.67mmol) in water-soluble (2mL), is added in the reaction mixture at 0 ℃ then with Sodium Nitrite.Reaction mixture was kept three days at 0 ℃.Reaction mixture is poured on ice, filters the product that collecting precipitation goes out then and wash with water.Vacuum-drying obtains title compound 800mg (75% yield).
MS (ESI) m/z 635 and 637[M+1] +
F) (2R, 2 ' R)-2,2 '-{ dithio-two [(2-methoxyl group [1,3] thiazole is [4,5-d] pyrimidine-5 also, 7-two bases) (methylene imine base)] } two (penta-1-alcohol)
Figure G2007800360541D00412
At 0 ℃, to be dissolved in the potassium hydroxide (210mg in the methyl alcohol (20mL), 3.75mmol) be added to (2R, 2 ' R)-2,2 '-{ dithio-is two, and [(2-chlorine [1,3] thiazole is [4,5-d] pyrimidine-5 also, 7-two bases) (methylene imine base)] } (795mg is 1.25mmol) in the solution in methyl alcohol (40mL) for two (penta-1-alcohol).Reaction mixture is spent the night 0 ℃ of stirring, steam methyl alcohol then.Resistates is poured in the ice, filtered then and collect the precipitation that forms.Filtrate is used ethyl acetate extraction.Organic phase merges resistates and the previous solid of collecting through dried over sodium sulfate and vacuum concentration, obtains title compound, and it just is used for next step without any being further purified.
MS(ESI)m/z?627[M+1] +。
G) 5-[7-{[(1R)-and 1-(methylol)] (methyl) amino }-[1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone-5-base dithio-also]-7-{[(1R)-and 1-(methylol-butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00421
Will from previous step obtain rough (2R, 2 ' R)-2,2 '-{ two [(the 2-methoxyl groups [1 of dithio-, 3] thiazole also [4,5-d] pyrimidine-5,7-two bases) (methylene imine base)] two (penta-1-alcohol) (1.25mmol) are dissolved in 1, in the 4-dioxane (25mL).Add concentrated hydrochloric acid (0.5mL) and water (0.5mL), then the mixture that forms is spent the night 45 ℃ of stirrings.The vacuum-evaporation dioxane is poured on resistates on ice then, so that make the product precipitation, collects product after filtration.Vacuum-drying obtains 590mg (two step yields 78%) title compound.
MS(ESI)m/z?599[M+1] +。
H) 5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol) butyl] (methyl) amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00422
Title compound is according to general method G, use 5-[7-{[(1R)-1-(methylol)] (methyl) amino }-[1,3] thiazole also [4,5-d] pyrimidine-2 (3H)-ketone-5-base dithio-]-7-{[(1R)-and 1-(methylol-butyl] amino } [1,3] thiazole also [4,5-d] pyrimidine-2 (3H)-ketone (0.10g, 0.167mmol), 2-chloro-5-[(1R)-the 1-chloroethyl] pyridine (embodiment 6g, 0.065g, 0.37mmol) and sodium borohydride (0.038g, 1.00mmol) preparation, obtain 0.060g (41% yield) title compound.
1H?NMR(CDCl 3)：δppm?8.56-8.38(m，1H)，7.87-7.73(m，1H)，7.28-7.26(m，1H)，4.86(q，1H)，4.75-4.62(m，1H)，3.76-3.55(m，3H)，3.03(s，3H)，1.70-1.63(m，3H)，1.53-1.45(m，2H)，1.26-1.21(m，2H)，0.95-0.88(m，3H)；
MS (ESI) m/z 440 and 442[M+1] +
Embodiment 8
Embodiment 8a
5-{[(1R)-and 1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also,With
Embodiment 8b
5-{[(1S)-and 1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also
Figure G2007800360541D00431
5-{[1-(3-fluorine pyridin-4-yl) ethyl from embodiment 3] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole also [4,5-d] non-enantiomer mixture of pyrimidine-2 (3H)-ketone (179mg) separates through preparation property HPLC, obtains the at first isomer that goes out of wash-out of 25mg:
1H NMR (DMSO-d 6): δ ppm 12.31 (br s, 1H), 8.51 (m, 1H), 8.38 (d, 1H); 7.62 (m, 1H); 6.97 (br s, 1H); 5.16 (q, 1H); 4.66 (t, 1H); 4.12 (m, 1H); (3.44-3.30 m, 2H are sheltered by the water signal), 1.66 (d, 3H), 1.61-1.27 (m, 3H), 0.84 (d, 3H), 0.74 (d, 3H); MS (ESI) m/z 424[M+1] +
And the isomer that goes out of the last wash-out of 45mg:
1H NMR (DMSO-d 6): δ ppm 12.35 (br s, 1H), 8.52 (d, 1H), 8.38 (d, 1H); 7.62 (dd, 1H); 7.12 (br s, 1H); 5.15 (q, 1H); 4.62 (t, 1H); 4.21 (m, 1H); (3.35-3.15 part is sheltered by the water signal for m, 2H), 1.65 (d, 3H), 1.63-1.29 (m, 3H), 0.88 (d, 3H), 0.85 (d, 3H); MS (ESI) m/z 424[M+1] +
The pharmacology screening
Material
The people CXXXC chemotactic molecule (hCX of reorganization 3CL1) and the reorganization Ro 24-7472/000-8 (IL-8 or hCXCL8) purchase the Inc. in PeproTech, UK.Have 2200 Ci/mmol specific activities reorganization [ 125I]-CXXXC chemotactic molecule (people) and [ 125I] hIL-8 purchase in Life Science Products, Inc., UK.Fluo4-AM purchases the Probes in Molecular, US.All other chemical reagent are analytical grades.
Cell
Complete people CX 3CR1 cDNA (GenBank accession number U20350) extracts from human brain mRNA (Superscript, Life Technologies), and connects (ligate) in pCR-BluntII TOPO carrier (In Vitrogen).Isolate the corresponding hCX of inset 3CR1 (insertcorresponding hCX 3CR1), and further subclone is in pcDNA3.1zeo.Use PlasmidMidi test kit (Qiagen) preparation plasmid DNA.The scheme that provides according to manufacturers then uses the Superfect transfection agents (Qiagen) will be at hCX 3The expression plasmid of CR1 is introduced human embryo kidney (HEK) and is suspended in (HEKS) 293 clones, and described clone contains and is useful on the chimeric G-Protein G of stably express α Qi5Carrier.Utilize Zeocin (500 μ g/mL) and Totomycin (hygromycin) (100 μ g/mL) to select to produce stable clone.For further application, cell is maintained among Dulbecco improvement Eagle substratum/Ham nutritional blend F12 (DMEM/F12), described DMEM/F12 contains Vit B6 (pyridoxine), and is supplemented with 10% (v/v) foetal calf serum, 2mM L-glutaminate, 100U/ml penicillin and 100mg/ml Streptomycin sulphate, 250 μ g/mL Zeocin and 100 μ g/mL Totomycin.
To in EMEM, cultivate available from the cell of the expressing human CXCR2 of AstraZeneca Charnwood, described EMEM contains Glutamax, and be supplemented with 10%FBS (from PAA, Austria), 1% non-essential amino acid (NEAA), 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates (PEST) and 500 μ g/mL Geneticin/G418.
Membrane prepare
With cell at 37 ℃, 5% CO 2Cultivate under the condition, and in the damping fluid that contains 10mM Tris-HCl pH7.4,5mM EDTA, 0.1mg/mL bacitracin (bacitracin), collect with the 60-80% fusion rate.With cell centrifugal 10 minutes with 300xg, to precipitate (pellet) resuspending then in collecting damping fluid (10mM Tris-HCl, pH 7.4,5mM ethylenediamine tetraacetic acid (EDTA) (EDTA) and 0.1mg/mL bacitracin) in, compile, use Dounce homogenizer (homogeniser) homogenate then.With homogenate centrifugal 10 minutes, use Ultra-Turrax T8 resuspending then in collecting damping fluid with 48000xg.With the film aliquots containig-80 ℃ of storages.As Harrington (1990, Anal.Biochem.186,285-287) described, in microtiter plate, determine protein concn.
Extracorporeal receptor is in conjunction with mensuration
2mL 96 deep-well plates (Beckman, Germany) in the cumulative volume in 1000 μ L/ holes carry out [ 125I] competition of CXXXC chemotactic molecule is in conjunction with research.Every hole contains that (50mMHepes-KOH, pH 7.4,10mM MgCl in measuring damping fluid 2, 1mM EDTA, 0.1% (w/v) gelatin) in 10pM [ 125I]-CXXXC chemotactic molecule and the film that is equivalent to the acceptor density of 1pM.(2 points/log unit) test compounds predissolves of ten kinds of concentration in DMSO, is added then to reach the ultimate density of 1% (v/v) DMSO.Begin to measure by adding film, hatch 24h at 25 ℃ then.Reaction stops in the following manner: through filtering fast with the pretreated Whatman GF/B of 0.3% polymine (polyethylimine) glass fibre filter, use Brandel receptors bind collector to wash with ice-cold damping fluid (10mM Hepes-KOH pH 7.4,500mM NaCl) subsequently.Add flicker cocktail type mixture (Scintillation cocktail), (Perkin Elmer determines radioactivity in USA) at Packard 2500TR liquid scintillation counter then.
In white clear bottom 96 hole Isoplate single part (singlicates) carry out [ 125I]-the hIL-8 competition is in conjunction with research, and final volume is 200 μ L, and every hole is contained in measuring damping fluid [50mM HEPES-KOH pH7.4,10mM MgCl 2, 1mM EDTA, 0.5% (w/v) gelatin] in 150pM[ 125I]-hIL-8 (specific activity 2200 Ci/mmol), the film-SPA goods that are equivalent to the 20pM acceptor density and 1.5mg SPA-pearl.Test compounds is as above handled.In the presence of the unlabelled hIL-8 of 500nM, determine non-specific binding.Agonist hIL-8 (concentration-response curve 3pM to 30nM) is as reference compound when each test.The peptide curve does not contain DMSO.Association reaction begins by adding 140 μ L film-SPA goods, then sample is hatched 4h in the room temperature lucifuge.With assay plate at liquid scintillation counter (Wallac
Figure G2007800360541D00451
TriLux 1450, from PerkinElmer, and USA) middle counting.
[ 35S] GTP γ S combination
Carry out at room temperature duplicate inhibitor ((2 concentration/log unit) are diluted among the DMSO (ultimate density 1%)) with 10 kinds of concentration in the clear bottom microtiter plate [ 35S] GTP γ S is in conjunction with research.To express hCX 3The film of CR1 acceptor (ultimate density 20 μ g protein/holes) adds together with the SPA pearl (ultimate density 1mg/ hole) that all is suspended in the GTP γ S binding buffer liquid (50mM Tris-HCl, 100mM NaCl, 0.1% gelatin, 15 μ g saponin(e/mL and 3 μ M GDP, room temperature pH 7.4).With film, SPA pearl and medicine preincubate 30 minutes, add 310pM CXXXC chemotactic molecule then and be used for maximal stimulation.Basis active (Basal activity) is defined as the activity of determining when not having the CXXXC chemotactic molecule to stimulate (GTP γ S binding buffer liquid).After extra 30 minutes, by add [ 35S] GTP γ S make ultimate density be 0.1nM and finally measure volume be 0.2mL begin the reaction.After 30 minutes, by stopping experiment with 2000rpm centrifugal 2 * 5 minutes (different directions), and at liquid scintillation counter (Wallac
Figure G2007800360541D00452
TriLux1450) determine radioactivity in.
The result
The typical CX of The compounds of this invention 3CR1 Ki value is in about scope of 0.1 to about 1000nM.CX 3Other of CR1 Ki is worth at about 0.1nM to the scope of about 500nM.CX 3Other of CR1 Ki is worth at about 0.1nM to the scope of about 25nM.The external hCX of final compound 3CR1 is shown in the table 1 in conjunction with measurement result.
Table 1.
The embodiment numbering K i(nM)
1 5.8
2 20
3 Not test *
4 18
5 Not test **
6 21.4
7 440
8a 97
8b 1.5
*) non-enantiomer mixture of embodiment 8a and 8b
*) do not obtain enough amounts to be used at external hCX 3CR1 tests in measuring
The compounds of this invention (R wherein 1Expression Me (at 5 sulfenyl Alkylpyridyls that contain branching) and corresponding reference compound (R wherein 1Expression H) comparing is more potent CX 3CR1 receptor antagonist and/or render a service relatively poor CXCR2 receptor antagonist.It is believed that this to CX 3The enhancing selectivity of CR1 receptor antagonism causes significant treatment benefit.

Claims (20)

1. the formula of free alkali form (I) compound or pharmaceutically acceptable salt thereof,
Figure FSB00000580244600011
Wherein:
R 1Be CH 3
R 2The expression halogen;
R 3Expression H or CH 3
R 4Expression H or CH 3
N represents 0,1 or 2.
2. the described compound of claim 1, wherein n represents 1.
3. the described compound of claim 1, wherein R 2Expression F or Cl.
4. the described compound of claim 1, wherein n represents 1; R 1Expression CH 3And R 2Expression F or Cl.
5. the described compound of claim 1, wherein pyridine connects with its 5 and has Cl 2 of pyridines.
6. the described compound of claim 1, wherein pyridine connects with its 2 and has F 5 of pyridines.
7. the described compound of claim 1, wherein pyridine connects with its 2 and has Cl 5 of pyridines.
8. the described compound of claim 1, wherein pyridine connects with its 2 and has F 3 of pyridines.
9. the described compound of claim 1, wherein pyridine connects with its 4 and has F 3 of pyridines.
10. the described compound of claim 1, wherein R 3Expression H.
11. the described compound of claim 1, wherein R 4Expression CH 3
12. the compound or pharmaceutically acceptable salt thereof of free alkali form, described compound is selected from:
5-{[(1S)-and 1-(5-chloropyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(5-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1R)-and 1-(3-fluorine pyridin-4-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(3-fluorine pyridine-2-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol)-3-methyl butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also;
5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-{[(1R)-and 1-(methylol) butyl] amino } [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also; And
5-{[(1S)-and 1-(6-chloropyridine-3-yl) ethyl] sulfenyl }-7-[[(1R)-and 1-(methylol) butyl] (methyl) amino] [1,3] thiazole [4,5-d] pyrimidines-2 (3H)-ketone also.
13. pharmaceutical composition, it comprises each described compound or pharmaceutically acceptable salt thereof in the claim 1 to 12, and is mixed with pharmaceutically acceptable diluent or carrier.
14. the compound or pharmaceutically acceptable salt thereof of each definition is preparing as CX in the claim 1 to 12 3The antagonist of CR1 acceptor is used for the treatment of or prevents purposes in the medicine of neurodegenerative disease, demyelinating disease, cardiovascular and cerebrovascular atheromatosis, peripheral arterial disease, rheumatoid arthritis, pulmonary disorder or pain.
15. the purposes of claim 14, wherein said pulmonary disorder are COPD or asthma.
16. the compound or pharmaceutically acceptable salt thereof of each definition is preparing as CX in the claim 1 to 12 3The antagonist of CR1 acceptor is used for the treatment of or prevents purposes in the medicine of multiple sclerosis.
17. the compound or pharmaceutically acceptable salt thereof of each definition is preparing as CX in the claim 1 to 12 3Purposes in the medicine that the antagonist of CR1 acceptor is used for the treatment of or prevention of arterial is atherosis, described medicine treat with the danger that reduces plaque rupture and atherosclerosis thrombosis incident by the composition that changes patch or prevention of arterial atherosis.
18. the compound or pharmaceutically acceptable salt thereof of each definition is preparing as CX in the claim 1 to 12 3Purposes in the medicine that the antagonist of CR1 acceptor is used for the treatment of or prevention of arterial is atherosis, described medicine by prevent and/or reduce new atherosclerotic lesions or patch formation and/or by the progress that prevents or slow down existing damage and patch treat or prevention of arterial atherosis.
19. the compound or pharmaceutically acceptable salt thereof of each definition is preparing as CX in the claim 1 to 12 3The antagonist of CR1 acceptor be used for the treatment of or the medicine of preventing apoplectic or transience brain injury in purposes.
20. the method for formula (I) compound or pharmaceutically acceptable salt thereof of each definition in the preparation claim 1 to 11, described method comprises:
A) make the reaction of formula (II) compound and formula (III) compound,
Formula (II) compound:
Figure FSB00000580244600031
R wherein 3And R 4Suc as formula defining in (I);
Formula (III) compound:
R wherein 1, R 2Define suc as formula (I) is middle with n, and L 1The expression leaving group; Or
B) make formula (IV) compound hydrolysis,
Figure FSB00000580244600041
R wherein 1, R 2, R 3, R 4Define suc as formula (I) is middle with n;
And randomly, formula (I) compound or its another kind of salt of gained is converted into its pharmacologically acceptable salt; Or formula (I) compound of gained is converted into another compound of formula (I) compound; And randomly, formula (I) compound of gained is converted into its optical isomer.
CN2007800360541A 2006-09-29 2007-09-27 Novel 5, 7-disubstituted [1, 3 ] thiazolo [4, 5-d] pyrimidin-2 (3h)-one derivatives and their use in therapy Active CN101516893B (en)

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