CN101611009A

CN101611009A - Quinoline

Info

Publication number: CN101611009A
Application number: CNA2007800346135A
Authority: CN
Inventors: 瑟格弗雷德·赫吉米; 凯文·迈克波里德; 阿布德尔玛德基德·K·熙熙; 一兰纳耶·卡尼可卡; 王颖; 斯蒂芬·李奥纳多·海耶斯; 玛丽-皮尔勒·谷曼德; 盖伊·思维格尼; 丹尼尔·杜马斯; 朱莉安·史密斯
Original assignee: CHRONOGEN Inc
Current assignee: CHRONOGEN Inc
Priority date: 2006-07-25
Filing date: 2007-07-25
Publication date: 2009-12-23
Also published as: RU2009106461A; WO2008014602A1; CA2658793A1; AU2007280984A1; JP2009544631A; EP2079699A1

Abstract

The present invention relates to new quinoline as having active CLK-1 inhibitor.More particularly, the CLK-1 inhibitor of the present invention compound that is formula (A).The invention still further relates to the medicine that comprises this compound and be used for preventing and/or treating the disease of suppress being benefited from CLK-1 or its its related indication method.

Description

Quinoline

Technical field

The present invention relates to new quinoline as the CLK-1 inhibitor.More particularly, the present invention relates to new quinoline or its pharmacologically acceptable salt or prodrug, comprise its pharmaceutical composition, and be used for preventing and/or treating illness or its related indication method of being benefited from the CLK-1 inhibition as the CLK-1 inhibitor.

Background technology

The effect of clk-1 and CLK-1

Clk-1 genes encoding 21kDa mitochondrial protein, it is guarded in the middle of eukaryote, and the sequence identity between mouse and the human sequence is 85%, and the homologous sequence of Caenorhabditis elegans (Caenorhabditiselegans) is 50%.CLK-1 participates in the hydroxylation of one of biosynthetic final step of ubiquinone and responsible DMQ 9 (DMQ).Wherein but CLK-1 is the ubiquinone (Q) of the synthetic detection limit of inactive mutant strain, but gathers DMQ people such as (, 2001) Miyadera.

Q has many functions in cell.It involves the electron transport of inner mitochondrial membrane, plasma membrane and lysosome membrane.The transduce cofactor in hole (MPTP) of its plastosome perviousness that still to be some cellular enzymes (dihydroorate dehydrogenase for example, it is that biological nucleic acid is synthetic necessary) and other protein and mixture separate even albumen (UCP) and adjusting apoptosis as the plastosome of regulating metabolic heat production.

DMQ is the quinones that can transport electronics, although it is so effective to be not so good as ubiquinone.The mouse embryo who has wherein rejected clk-1 homologue (mclk-1) also gathers DMQ, and can not survive.People such as (, 2001) Levavasseur, but the mouse heterozygote of mclk-1 has shown the lifetime phenotype that increases.The transgene expression of CLK-1 has been rescued the mortality phenotype in rejecting mouse, and allows synthetic people such as (, 2004) Nakai of ubiquinone.Be enjoyably, the heterogenous expression of Caenorhabditis elegans CLK-1 has effect in slow growing coq7 (yeast clk-1) yeast mutant, and rescues the slow phenotype of yeast growth (people such as Ewbank, 1997).In a word, these observations show that CLK-1 is the unique DMQ hydroxylase in being studied organism, and not for the active function compensation mechanism of CLK-1.

In Caenorhabditis elegans, the active reduction of CLK-1 also changes the increase of lifetime into.Lifetime is the accumulation phenotype of in worm physiology various variations being integrated, and it is responsive to ambient signal and transgenation.Thereby the condition that prolongs the lifetime is considered to useful usually.For example, favourable reparation to the balance of damage can be by increasing the lifetime self performance people such as (, 2001) Hekimi.

The age associated conditions

Age-related disease is meant the disease that increases along with the increase sickness rate at age and/or morbidity.These diseases comprise for example cancer, cerebrovascular disease (palsy), neurodegenerative disease, pneumonia, respiratory system disease, sacroiliitis, heart trouble, diabetes, dysaudia, visual disorder and ephrosis.

Active and the age associated conditions of CLK-1

CLK-1 is suppressed at the effect that has the similar prolongation lifetime in mouse and the nematode.This has shown the causal relation between CLK-1 biological activity and physiology weathering process consumingly.Generally accepted be physiology aging be the Hazard Factor of numerous disease (so-called age dependence disease).Many these diseases are fatal and have limited the lifetime.Shown when mouse CLK1 (mCLK1) protein level mclk1+/-reduce half people such as (, 2001) Levavasseur in the heterozygote, it has increased lifespan, intermediate value lifetime and the maximum lifespan of these animals in three different experiments.(129SV/j, C57BL/6 and 129SV/j x Balb/c) (people such as Liu, 2005) are carried out in each experiment under different genetic background.Animal under these backgrounds is known to die from various age dependence diseases, and its pattern is different (people such as Blackwell, 1995 under each background; People such as Cosgrove, 1978; People such as Frith, 1983; People such as Smith, 1973).In three all experiments, most of wild-type animal and mutant animals are dead in the relative short period, this be genotype (mclk1+ /+or mclk1+/-) typical case's performance people such as (, 2005) Liu.The fact that the fact that the Mclk1 minimizing is worked under different genetic background and maximum lifespan and intermediate value lifetime are increased shows that all or most mortality reason must be by this minimizing part ground compacting.In fact, when the reason of single lifetime is subjected to genetic manipulation such as cancer and influences, the lifetime only minimally prolong or more prolong (people such as Matheu, 2004; Miller, 2005).Sickness rate (Bartke, 2005 of the operation that reduces such as heat restriction and tethelin signal transduction by contrast, (its known make physiology is aging to slow down) reduction age dependence disease; People such as Berrigan, 2002; People such as Hursting, 2004).In addition, wherein weathering process is able to that promoted animal has shown that the disease morbidity increases and disease is early sent out (people such as Fenton, 2004; People such as Takeda, 1997).These are observed together with clk-1/mclk1 the evolution conservative of lifetime people such as (, 2005) Liu are shown that reducing the mCLK1 activity makes that physiology is aging to slow down, so the inhibition of CLK-1 can postpone the morbidity and the seriousness of age dependence disease.The known physiology that slows down is aging to be to reduce the sickness rate of various diseases and the best way (Finkel, 2005 of seriousness; Miller, 2005).

Therefore, the chemical that can be used as the CLK-1 inhibitor by use suppresses CLK-1 and causes prevention or reduce age-related disease.

Quinoline

The compound of quinoline type is disclosed among CA 2.493.536A1, US2006/0074104A1 and the WO 2004/087160A1, be used for the treatment of, improve and/or prevent the neurological patient's condition, particularly relevant or by its promoted those patient's condition with oxidative stress.The neurological illness of taking into account comprises any cognitive impairment such as any patient's condition early stage or the mild cognitive damage or the loss of memory of causing.

Although it is useful oxidative stress conditioning agent that above-mentioned quinoline has shown, it is said that they mainly bring into play its curative effect by the metal-chelating effect.There is not the prior art document to show that similar compound can be used for preventing and/or treating the age associated conditions of being benefited from CLK-1 suppresses so far.

In this respect, the inventor has developed new gang and has and suppress the active compound of CLK-1.These compounds have activity to age associated conditions and ischemical reperfusion injury and inflammatory condition.

Summary of the invention

Therefore, the purpose of this invention is to provide the quinoline of new formula A, it has CLK-1 and suppresses active.

More particularly, the present invention relates to compound as the formula A that gives a definition, or its pharmacologically acceptable salt or its prodrug;

X=H wherein, methyl or halogen, and substituent R ₁And R ₂Be defined as follows described:

A) work as R ₂During for hydrogen, R then ₁Be H or halogen, perhaps R ₁Be selected from: amino (3, the 4-Dimethoxyphenyl) methyl, 2-hydroxy phenyl, 3-hydroxy phenyl, 4-hydroxy phenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2-methoxypyridine-3-base and 2-acetamido phenyl;

Perhaps R ₁For-CH (R ₃) NR ₄R ₅, wherein

R ₄=H or C ₁-C ₄Alkyl;

R ₅=H or

And R ₆Be selected from: C ₁-C ₆Alkyl, C ₅-C ₆Aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenyl methyl; With

R ₃Be selected from: H, methyl, phenyl, benzyl, the 2-chloro-phenyl-, 2-p-methoxy-phenyl, 4-chloro-phenyl-, 4-aminomethyl phenyl, the 4-isopropyl phenyl, 3-hydroxy phenyl, 4-hydroxy phenyl, the 4-p-methoxy-phenyl, 4-cyano-phenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, the 2,4 dichloro benzene base, 3,4-Dimethoxyphenyl, 4-methoxycarbonyl phenyl, 3-methoxycarbonyl phenyl, N-methyl-benzamide base, N-(3-methoxy-propyl) benzoylamino, N, N-dimethyl benzamide base, 4-(morpholine-4-base carbonyl) phenyl, 4-(pyridine-2-yl) phenyl, 4-(1H-pyrazol-1-yl) phenyl, pyridine-2-base, pyridin-3-yl, pyridin-4-yl, 6-(morpholine-4-yl) pyridin-3-yl, the 2-furyl, 3-furyl, 2-(morpholine-4-yl) pyridin-3-yl, 2-methoxypyridine-3-base, 4-methoxypyridine-3-base, 2-thienyl, 2-butyl-1H-imidazol-4 yl, quinoline-3-base, quinolyl-4, oxine-2-base, 1H-indol-3-yl, 1H-indoles-4-base and 1H-indoles-7-base;

Perhaps R ₁For

And R ₇For-(CH ₂) n-R ₈, R wherein ₈Be the C that is randomly replaced by 1 or 2 methoxyl group ₅-C ₆Aryl, and n=0 or 1; Perhaps

B) as X and R ₁When all representing hydrogen atom, R then ₂Be selected from: pyridine-2-base formamido-, pyridine-2-yl acetamide base, 3-hydroxy phenyl acetamido, 4-hydroxy phenyl acetamido, ((2-p-methoxy-phenyl) amino) methyl, ((3-p-methoxy-phenyl) amino) methyl, ((3-methoxy-benzyl) amino) methyl, 2-thienyl acetamido and ((2-thienyl methyl) amino) methyl;

Restricted condition is that each compound of formula A is not one of compound pointed in the annex 1.

The compound of formula A of the present invention is the form of one of its optically active isomer such as enantiomorph when comprising at least one asymmetric center, perhaps be to comprise for example its form of mixtures of racemoid.

The invention still further relates to and be used for suppressing the active method of CLK-1 at cell, this method may further comprise the steps: a) providing wherein needs to suppress the active cell of CLK-1, b) makes compound or its pharmacologically acceptable salt or the prodrug of this cells contacting as the formula (B) given a definition;

Wherein X, R ₁And R ₂Has definition as hereinbefore;

The compound of formula (B) is the form of one of its optically active isomer or the form of its mixture when comprising at least one asymmetric center; With

C) the CLK-1 activity in the mensuration cell.

The invention still further relates to and be used for preventing and/or treating illness or its related indication method of being benefited animal from CLK-1 suppresses, this method may further comprise the steps: a) identify the animal that suffers from the illness of being benefited from CLK-1 suppresses; With

B) to compound, its pharmacologically acceptable salt or the prodrug of described animal to the formula of using (B).

The invention still further relates to pharmaceutical composition, it comprises formula A compound of the present invention, its pharmacologically acceptable salt or prodrug, and at least a pharmaceutically useful carrier.

The invention still further relates to pharmaceutical composition, it comprises the A of formula as defined above of pharmacy effective dose or compound, its pharmacologically acceptable salt or the prodrug of formula B, and at least a pharmaceutically useful carrier, to reduce effectively and/or completely or partially to suppress the CLK-1 activity.

Description of drawings

Fig. 1: show with the CLK-1 inhibitor and handle the back HPLC curve that DMQ reduces in the quinone curve.Mouse macrophage (RAW264.7) a) non-active compound or b) active compound 135 (1 μ M) handled 24 hours, carried out cytolysis then and with hexane/extraction using alcohol quinone.Sample is used the methanol/ethanol gradient elution on the HPLC that has the UV detector, thereby measure the level of ubiquinone (Q) and precursor DMQ 9 (DMQ) thereof.

Fig. 2: be presented at and use a) DMSO or b) the HPLC curve (Q: ubiquinone, DMQ: DMQ 9) of relative quinone peak value in the RAW264.7 cell that the compound 7 of 10 μ M is handled.

Fig. 3: be presented at use a) 1 μ M's or b) the HPLC curve (Q: ubiquinone, DMQ: DMQ 9) of relative quinone peak value in the RAW264.7 cell handled of the compound 23 of 3 μ M.

Fig. 4: be presented at use a) 5 μ M's or b) the HPLC curve (Q: ubiquinone, DMQ: DMQ 9) of relative quinone peak value in the RAW264.7 cell handled of the compound 69 of 10 μ M.

Fig. 5: be presented at use a) 5 μ M's or b) the HPLC curve (Q: ubiquinone, DMQ: DMQ 9) of relative quinone peak value in the RAW264.7 cell handled of the compound 47 of 10 μ M.

Fig. 6: be presented at use a) 5 μ M's or b) the HPLC curve (Q: ubiquinone, DMQ: DMQ 9) of relative quinone peak value in the RAW264.7 cell handled of the compound 53 of 10 μ M.

Fig. 7: the specificity of CLK-1 inhibitor.7 pairs of a) mouse CLK-1 (JF496-mclk1 is for lifetime measurement system) and b) effects of bacterium DMQ hydroxylase UbiF (JF496-ubiF is for lifetime measurement system) of compound.Express a) mouse CLK-1 and b) the JF496 bacterium of bacterium DMQ-hydroxylase UbiF handled 5 hours with compound 7.Extract quinone and use HPLC to analyze.The HPLC curve shows that the active combined thing 7 of CLK-1 suppresses, because the synthetic increase that reduces and follow DMQ 9 (DMQ) of ubiquinone (Q).In contrast to this, the UbiF activity is unaffected, and Q is the main quinone material that is detected.This shows that compound 7 has specific CLK-1 and suppresses active.

Fig. 8: the specificity of CLK-1 inhibitor.52 pairs of a) mouse CLK-1 (JF496-mclk1 is for lifetime measurement system) and b) effects of bacterium DMQ hydroxylase UbiF (JF496-ubiF is for lifetime measurement system) of compound.Express a) mouse CLK-1 and b) the JF496 bacterium of bacterium DMQ-hydroxylase UbiF handled 5 hours with compound 52.Extract quinone and use HPLC to analyze.The HPLC curve shows that the active combined thing 52 of CLK-1 suppresses, because the synthetic increase that reduces and follow DMQ 9 (DMQ) of ubiquinone (Q).In contrast to this, the UbiF activity is unaffected, and Q is the main quinone material that is detected.This shows that compound 52 has the special inhibition activity of specific CLK-.

Fig. 9: the ROS reduction effect of CLK-1 inhibitor (7 and 135).(A) show in mouse macrophage form about the quinone curve of CLK-1 inhibitor; (B) handle with fluorescent marker DCF with the cell of CLK-1 inhibitor preincubation, it produces green fluorescence when by cell ROS oxidation.In cell, use H ₂O ₂Induce ROS, and use the observed reduction of CLK-1 inhibitor to represent with respect to the percentage ratio of contrast; (C) use the COMET experimental measurement of fluorescence afterbody of the DNA that spills from nucleus by ROS inductive dna damage.Reuse H ₂O ₂Induce ROS, and data with the cell with afterbody compared with the control percentage ratio represent.Shown the minimizing (% of total cell) that under the condition that the CLK-1 inhibitor exists, shows the cell of comet.

Figure 10: the effect of

compound

7 or 52 pairs of serum creatinine level in the ischemia-reperfusion rat model.

Figure 11: the effect of

compound

7 or 52 pairs of blood urea nitrogens (BUN) level in the ischemia-reperfusion rat model.

Figure 12: the effect of

compound

7 or 52 pairs of creatinine clearances in the ischemia-reperfusion rat model.

Figure 13: the effect of

compound

7 or 52 pairs of urine protein concentration (g/L) in the ischemia-reperfusion rat model.

Figure 14: the effect of 7 pairs of lipoxygenases of compound (LPO) level in the rat of lipopolysaccharides (LPS) inductive injury of lung.

Figure 15: the effect of 7 pairs of protein contents of compound in the rat of inducing injury of lung with LPS.

Figure 16: the effect of 7 pairs of total cell levelss of compound and neutrophilic granulocyte level in the rat of inducing injury of lung with LPS.

Figure 17: the effect of 7 pairs of TNF-alpha levels of compound in the rat of inducing injury of lung with LPS.

Figure 18: the effect that the compound 138 of the multiple dosage of measuring by ELISA in the hAPP751SL transgenic mice in the TBS fraction of usefulness is handled solvable A β 1-40.

Figure 19: the effect that the compound 138 of the various dosage of measuring by ELISA in the hAPP751SL transgenic mice in the TBS fraction of usefulness is handled solvable A β 1-42.

Figure 20: the effect that the compound 138 of the various dosage of measuring by ELISA in the hAPP751SL transgenic mice in Triton X-100 fraction of usefulness is handled solvable A β 1-40.

Figure 21: the effect that the compound 138 of the various dosage of measuring by ELISA in the hAPP751SL transgenic mice in Triton X-100 fraction of usefulness is handled solvable A β 1-42.

Figure 22: the compound treatment of the multiple dosage of measuring by ELISA in the hAPP751SL transgenic mice in the FA fraction of usefulness is to the effect of mating type A β 1-40.

Figure 23: the compound treatment of the multiple dosage of measuring by ELISA in the hAPP751SL transgenic mice in the FA fraction of usefulness is to the effect of mating type A β 1-42.

Detailed Description Of The Invention

The present invention has obtained beyond thought discovery,, comprises the noval chemical compound classification such as the quinoline of undefined formula A that is, and it shows CLK-1 and suppresses active.

Definition

Officinal salt used herein is intended to comprise any salt of the compounds of this invention, and this salt is suitable for contacting people and zootic tissue and matches without unsuitable toxicity, excitant or allergy and with rational risk/benefit ratio. Officinal salt is (Pharmaceutical Salts Properties well known in the art, Selection, and Use, Stahl, P.Heinrich/Wermuth, Camille G. (editor), 2002.Monograph-ISBN 3-906390-26-8-Verlag Helvetica Chimica Acta, Zurich). Officinal salt can in the end separate with the purifying the compounds of this invention during original place preparation, perhaps the free alkali functional group by making the compounds of this invention and suitable acid reaction preparation or the free acid functional group by making the compounds of this invention prepare with suitable alkali reaction individually, include but not limited to hydroxide, carbonate or the bicarbonate of pharmaceutically useful metal cation. Pharmaceutically useful acid-addition salts includes but not limited to acetate, adipate, alginates, citrate, aspartate, benzoate, benzene sulfonate, butyrate, camphorate, camsilate, digluconate, glycerophosphate, half sulfonate, enanthate, caproate, fumarate, hydrochloride, hydrobromate, hydriodate, 2-isethionate, lactate, maleate, mesylate, nicotinate, the 2-naphthalene sulfonate, oxalates, embonate, pectate, persulfate, 3-phenylpropionic acid salt, picrate, Pivalate, propionate, succinate, tartrate, rhodanate, nitrate, sulfate, disulfate, phosphate, acid phosphate, glutamic acid, bicarbonate, tosilate and hendecane hydrochlorate. In addition, comprising the group of basic nitrogen can be by quaternized such as following material: alkyl halide is chloride, bromide and the iodide of methyl, ethyl, propyl group and butyl for example; Dialkylsulfates is the sulfuric ester of dimethyl, diethyl, dibutyl and diamyl for example; Long-chain halide is chloride, bromide and the iodide of decyl, lauryl, myristyl and stearyl for example; With the aryl alkyl halide bromide of benzyl and phenylethyl for example. Officinal salt also includes but not limited to the cationic salt based on alkali metal or alkaline-earth metal, such as salt of aluminium, calcium, lithium, magnesium, potassium, sodium etc.

Pro-drug used herein refers to such compound, this compound in vivo during administration by metabolism or otherwise be converted into the compound of biology, pharmacy or the acology activity form of parent compound, for example by in blood, being hydrolyzed. In order to obtain pro-drug, thereby being modified reactive compound, pharmaceutically active compound will again be generated by metabolic process. Pro-drug can be designed to change metabolic stability or the K+transport (comprise and improve bioavilability) of medicine, shelters side effect or toxicity, improves further feature or the character of taste or the change medicine of medicine. Rely on the knowledge of the interior pharmacokinetics process of body and drug metabolism, those skilled in the art can design the pro-drug of this compound when learning pharmaceutically active compound.

The metabolin that the body internal strain of the compound of through type A or B forms has also been contained in the present invention. The compound that the term metabolin refers to formula A or B is via oxidation, reduction, hydrolysis or the compound that forms in conjunction with carrying out bio-transformation in the body.

Term alkyl used herein refers to saturated aliphatic group, comprises straight chained alkyl, branched alkyl and cycloalkyl (alicyclic) group.

Term aryl used herein comprises 5 and 6 yuan of monocyclic aromatic bases, and it can comprise 0-4 hetero atom (S, N, O), for example is unsubstituted or substituted benzene, pyrroles, furans, thiophene, imidazoles ， oxazole, thiazole, triazole, pyrazoles, pyridine, pyrazine, pyridazine and pyrimidine etc.

Term halogen used herein refers to F, Cl, Br or I.

Term optical isomer used herein refers to any isomeric form of compound required for protection, such as, but not limited to enantiomer, and diastereoisomer, racemate or its mixture.

It is the bioactive effectively and to host or the avirulent mounting medium of patient of formula A compound that pharmaceutically useful carrier refers to not disturb the active component of composition. In addition, this carrier advantageously has the compound of the minimum possibility that the immune system that is treated the experimenter repels. Suitable carrier is those skilled in the art's general knowledge and describes in detail no longer in addition.

Term treatment used herein refers to a kind of method, and by the method, the illness of being benefited from CLK-1 suppresses or its related symptoms are alleviated or be completely eliminated. For example, can think described treatment can be for example by with suitable dosage to be used in the suitable dosage form compound of the present invention to the excessive risk with disease/or the early stage patient of disease alleviate or eliminate a disease fully or its related symptoms.

Term prevention used herein refers to a kind of method, and by the method, illness or its related symptoms include but not limited to the age associated conditions, are obstructed, postpone or prevent.

The illness that statement used herein is benefited from CLK-suppresses refers to illness or its related symptoms known or that expection is benefited from CLK-1 suppresses. More particularly, this statement refers to that when CLK-1 is suppressed illness or its related symptoms are alleviated or eliminate. Even more particularly, this statement refers to but is not limited to inflammatory condition, by the illness that damage caused or increased the weight of of oxidative stress and/or free yl induction, comprises disease such as anoxic/oxygen enrichment damage and the ischemia/reperfusion injury of ROS mediation. The age associated conditions has also been contained in this statement.

Statement used herein age associated conditions refers to the illness that increases along with the increase incidence of disease at age and/or illness rate include but not limited to angiocardiopathy, atherosclerotic for example, coronary artery disease and palsy; Peripheral vascular disease; Metabolic disorder, such as type ii diabetes, dyslipidemias mass formed by blood stasis and hypertriglyceridemia; Cancer, such as cutaneum carcinoma, papilloma and age-dependent cancer; Ischemia/reperfusion injury, the ephrosis of inducing such as kidney, heart, cerebral ischemia and pneumoradiography; Inflammation; Neurodegenerative disorders and dementia, such as Alzheimer disease, parkinsonism, Huntingtons chorea, memory disorders and mental disease; Bladder and kidney disorders, such as ephritis, ephrosis, end-stage renal disease (ESRD); Diabetic syndrome, such as DPN, the poor and retinopathy of wound healing; Illness in eye, such as AMD, scheroma, glaucoma, retinitis pigmentosa and cataract; Lung and respiratory disorder are such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF); The illness of flesh and bone, such as inflammatory arthritis, gout and osteoporosis; And skin conditions, such as cutaneum carcinoma, and skin aging such as light aging.

Term inflammation used herein refers to be replied by the localised protection that the damage of tissue or destruction cause; it is enough to destroy, weaken or block harmful substance and damaged tissues; be characterised in that the acute form by the standard supervention representations of events of pain, heating, rubescent, swelling and afunction; and involve serial complicated event in histology; comprise parteriole, capilary and veinlet expansion; permeability and blood flow increase; the fluid that comprises plasma proteins oozes out, and leukocytoplania enters struvite focus. Be further characterized in that a large amount of release TNF-α.

Statement ischemia/reperfusion injury used herein refers to organize or organ is worked as the patient's condition that loses blood flow and suffer, and the insufficient institute owing to nutrition and oxygen causes mainly. Reperfusion injury refers to the tissue damage that suffers when when the ischemic that usually surpasses about ten minutes recovers blood flow after the period. Ischemia and reperfusion can cause the tissue that experiences this process is caused serious or fatal damage.

Quinoline of the present invention is the compound with following general formula (A) structure, or its officinal salt or its pro-drug:

X=H wherein, methyl or halogen, and substituent R₁And R₂Be defined as follows described:

A) work as R₂During for hydrogen, R then₁Be H or halogen, perhaps R₁Be selected from: amino (3,4-Dimethoxyphenyl) methyl, 2-hydroxy phenyl, 3-hydroxy phenyl, 4-hydroxy phenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2-methoxypyridine-3-base and 2-acetamido phenyl;

Perhaps R₁For-CH (R₃)NR ₄R ₅, wherein

R ₄=H or C₁-C ₄Alkyl;

R ₅=H or

Perhaps R ₁For And R ₇For-(CH ₂) n-R ₈, R wherein ₈Be the C that is randomly replaced by 1 or 2 methoxyl group ₅-C ₆Aryl, and n=0 or 1; Perhaps

The compound that comprises the formula of the present invention (A) of at least one asymmetric center is the form of one of its enantiomorph or the form of its mixture.

More preferably, the compound of formula (A) is such compound, wherein R ₄Be H or methyl, R ₆Be selected from methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, phenyl, benzyl, dimethylaminomethyl, phenylethyl, cyclohexyl, diphenyl methyl, pyridine-2-base and pyridin-3-yl, other substituting group has identical meanings as defined above.

According to another preferred version, the compound of formula (A) is selected from following:

N-((3, the 4-Dimethoxyphenyl) (oxine-7-yl) methyl) ethanamide 2,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl) benzamide 3,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl)-N-methylacetamide 8,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl)-2,2-phenylbenzene ethanamide 11,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl)-2-phenyl-acetamides 12,

7-(amino (3, the 4-Dimethoxyphenyl) methyl)-5-chloroquinoline-8-alcohol 13,

N-(1-(5-chloro-oxine-7-yl)-2-phenylethyl) ethanamide 14,

N-((2-butyl-1H-imidazol-4 yl) (5-chloro-oxine-7-yl) methyl) ethanamide 15,

N-((5-chloro-oxine-7-yl) (2-methoxypyridine-3-yl) methyl) ethanamide 16,

N-((5-chloro-oxine-7-yl) (quinolyl-4) methyl) ethanamide 17,

N-((5-chloro-oxine-7-yl) (2-morpholine-4-yl pyridines-3-yl) methyl)-ethanamide 18,

N-((5-chloro-oxine-7-yl) (quinoline-3-yl) methyl) ethanamide 19,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl) methyl benzoic acid ester 21,

3-((acetylamino) (5-chloro-oxine-7-yl) methyl) methyl benzoic acid ester 22,

N-((5-chloro-oxine-7-yl) (4-cyano-phenyl) methyl) ethanamide 23,

N-((5-chloro-oxine-7-yl) (4-pyridine-2-base phenyl) methyl) ethanamide 24,

N-((5-chloro-oxine-7-yl) (4-(1H-pyrazol-1-yl) phenyl) methyl) ethanamide 25,

N-((5-chloro-oxine-7-yl) (4-hydroxy phenyl) methyl) ethanamide 26,

N-((5-chloro-oxine-7-yl) (3-hydroxy phenyl) methyl) ethanamide 27,

N-((5-chloro-oxine-7-yl) (6-methoxypyridine-3-yl) methyl) ethanamide 28,

N-((5-chloro-oxine-7-yl) (pyridine-2-yl) methyl) ethanamide 29,

N-((5-chloro-oxine-7-yl) (pyridin-4-yl) methyl) ethanamide 30,

N-((5-chloro-oxine-7-yl) (pyridin-4-yl) methyl) acetamide hydrochloride 31,

N-((5-chloro-oxine-7-yl) (pyridin-3-yl) methyl) ethanamide 32,

N-((5-chloro-oxine-7-yl) (pyridin-3-yl) methyl) acetamide hydrochloride 33,

N-((5-chloro-oxine-7-yl) (oxine-2-yl) methyl) ethanamide 34,

N-((5-chloro-oxine-7-yl) (6-morpholine-4-yl pyridines-3-yl) methyl)-ethanamide 35,

N-((5-chloro-oxine-7-yl) (1H-indol-3-yl) methyl) ethanamide 36,

N-((5-chloro-oxine-7-yl) (1H-indoles-4-yl) methyl) ethanamide 37,

N-((5-chloro-oxine-7-yl) (1H-indoles-7-yl) methyl) ethanamide 38,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl)-N, N-dimethyl-benzamide 39,

N-((5-chloro-oxine-7-yl) (4-(morpholine-4-base carbonyl) phenyl) methyl)-ethanamide 40,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl) pyridine-2-carboxamide 41,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl)-N-(3-methoxy-propyl)-benzamide 44,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl)-N-methyl-benzamide 45,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl)-N, N-dimethyl-G-NH2 46,

N-(1-(5-chloro-oxine-7-yl) ethyl) ethanamide 47,

N-((oxine-7-yl) methyl) ethanamide 48,

N-(2-furyl methyl)-oxine-7-base methane amide 53,

N-(2-thienyl methyl)-oxine-7-base methane amide 54,

N-(2-methoxy-benzyl)-oxine-7-base methane amide 55,

5-chloro-7-(3-hydroxy phenyl) quinoline-8-alcohol 57,

5-chloro-7-(4-hydroxy phenyl) quinoline-8-alcohol 58,

N-(2-(5-chloro-oxine-7-yl) phenyl) ethanamide 59,

5-chloro-7-(2-methoxypyridine-3-yl) quinoline-8-alcohol 61,

5-chloro-7-pyridin-4-yl quinoline-8-alcohol hydrochloride 62,

N-(3, the 4-Dimethoxyphenyl)-oxine-7-base methane amide 63,

N-(3-p-methoxy-phenyl)-oxine-7-base methane amide 64,

N-(3-methoxy-benzyl)-oxine-7-base methane amide 65,

N-(3, the 4-dimethoxy-benzyl)-oxine-7-base methane amide 66,

N-(2-p-methoxy-phenyl)-oxine-7-base methane amide 67,

N-(pyridin-3-yl methyl)-oxine-7-base methane amide 68,

N-(oxine-2-yl) pyridine-2-base methane amide 69,

N-(oxine-2-yl)-2-pyridine-2-yl acetamide 70,

2-(3-hydroxy phenyl)-N-(oxine-2-yl) ethanamide 71,

2-(4-hydroxy phenyl)-N-(oxine-2-yl) ethanamide 72,

N-(oxine-2-yl)-2-(2-thienyl) ethanamide 73,

2-(((3-methoxy-benzyl) amino) methyl) quinoline-8-alcohol 74,

2-(((3-p-methoxy-phenyl) amino) methyl) quinoline-8-alcohol 75,

2-(((2-thienyl methyl) amino) methyl) quinoline-8-alcohol 76,

2-(((2-p-methoxy-phenyl) amino) methyl) quinoline-8-alcohol 77,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] ethanamide 78,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl] ethanamide 79,

N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl] ethanamide 80,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl] butyramide 81,

N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl] butyramide 82,

N-[(8-hydroxyquinoline-7-yl) (4-aminomethyl phenyl) methyl]-3-Phenylpropionamide 92,

The N-[(2-chloro-phenyl-) (oxine-7-yl) methyl]-3-Phenylpropionamide 93,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl]-3-methylbutyryl amine 94,

N-[(8-hydroxyquinoline-7-yl) (phenyl) methyl]-3-methylbutyryl amine 110,

N-[(8-hydroxyquinoline-7-yl) (4-p-methoxy-phenyl) methyl]-3-Phenylpropionamide 111,

N-[(3, the 4-Dimethoxyphenyl) (oxine-7-yl) methyl]-3-methylbutyryl amine 112,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl]-3-methylbutyryl amine 113,

The N-[(4-chloro-phenyl-) (oxine-7-yl) methyl] cyclohexane carboxamide 121,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] propionic acid amide 122,

N-[(3, the 4-Dimethoxyphenyl) (oxine-7-yl) methyl]-3-Phenylpropionamide 124,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] valeramide 125,

N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl] valeramide 126,

N-[[4-(diethylamino) phenyl] (oxine-7-yl) methyl] valeramide 127,

N-[(8-hydroxyquinoline-7-yl) (4-p-methoxy-phenyl) methyl] cyclohexane carboxamide 128,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] butyramide 129,

N-[(8-hydroxyquinoline-7-yl) (4-aminomethyl phenyl) methyl] cyclohexane carboxamide 130 and

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl] cyclohexane carboxamide 131.

According to preferred version, the prodrug of the compound of formula of the present invention (A) is equivalent to wherein the hydroxyl on 8 of quinoline moiety by the monobasic compound of following group:

Wherein M is selected from Li, Na, Ca and K.

According to another preferred version, dihydrogen phosphoric acid ester prodrug defined above is the form of its hydrochloride or dihydrochloride.

Preferred prodrug of the present invention is as follows:

((7-((acetylamino) (3, the 4-Dimethoxyphenyl) methyl)-5-chloroquinoline-8-yl) oxygen base) methyl dihydrogen phosphoric acid ester 5,

((7-((acetylamino) (3, the 4-Dimethoxyphenyl) methyl)-5-chloroquinoline-8-yl) oxygen base) methyl phosphorodithioate disodium 6,

((7-((acetylamino) (2-furyl) methyl)-5-chloroquinoline-8-yl) oxygen base) methyl dihydrogen phosphoric acid ester 136,

((7-((acetylamino) (2-furyl) methyl)-5-chloroquinoline-8-yl) oxygen base) methyl phosphorodithioate disodium 137,

((7-((acetylamino) (3, the 4-Dimethoxyphenyl) methyl)-5-chloroquinoline-8-yl) oxygen base) methyl dihydrogen phosphoric acid ester dihydrochloride 138 and

((7-((acetylamino) (3, the 4-Dimethoxyphenyl) methyl)-5-chloroquinoline-8-yl) oxygen base) methyl dihydrogen phosphoric acid ester hydrochloride 139.

As mentioned above, compound of the present invention can exist asymmetric center or chiral centre.The multiple optically active isomer and composition thereof of the compound of formula (A) has been contained in the present invention.For example, the independent enantiomorph of The compounds of this invention from the commercially available parent material that comprises asymmetric center or chiral centre synthetic make or mixture by the preparation enantiomeric compounds then by well known to a person skilled in the art that method for splitting makes.The example of these method for splitting is a) racemic mixture of enantiomorph to be attached on the chiral auxiliary(reagent), by recrystallization or the separating obtained diastereomer of chromatography, and the optically-active pure products separated or b from auxiliary agent) the direct mixture of dissociated optical enantiomorph on chiral chromatographic column.

The invention still further relates to and be used for suppressing the active method of CLK-1 at cell, this method may further comprise the steps:

A) provide and wherein need to suppress the active cell of CLK-1,

B) make the compound of this cells contacting formula (B):

X=H wherein, methyl or halogen, and substituent R ₁And R ₂As give a definition:

I) work as R ₂During for hydrogen, R then ₁Be H or halogen, perhaps R ₁Be selected from: amino (3, the 4-Dimethoxyphenyl) methyl, 2-hydroxy phenyl, 3-hydroxy phenyl, 4-hydroxy phenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2-methoxypyridine-3-base and 2-acetamido phenyl;

Perhaps R ₁For-CH (R ₃) NR ₄R ₅, wherein

R ₄=H or C ₁-C ₄Alkyl;

R ₅=H or

Perhaps R ₁For

And R ₇Be (CH ₂) n-R ₈, R wherein ₈Be the C that is randomly replaced by 1 or 2 methoxyl group ₅-C ₆Aryl, and n=0 or 1; Perhaps

Ii) as X and R ₁When all representing hydrogen atom, R then ₂Be selected from: pyridine-2-base formamido-, pyridine-2-yl acetamide base, 3-hydroxy phenyl acetamido, 4-hydroxy phenyl acetamido, ((2-p-methoxy-phenyl) amino) methyl, ((3-p-methoxy-phenyl) amino) methyl, ((3-methoxy-benzyl) amino) methyl, 2-thienyl acetamido and ((2-thienyl methyl) amino) methyl;

The compound of formula (B) is the form of one of its enantiomorph or the form of its mixture when comprising at least one asymmetric center; With

C) the CLK-1 activity in the mensuration cell.

Advantageously, be used for being selected from compound pointed in the table 1 at the compound of the cell inhibition active formula of CLK-1 (B).

Even more advantageously be to be used for being selected from following at the compound of the cell inhibition active formula of CLK-1 (B):

N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] ethanamide 7,

5-chloro-7-iodine quinoline-8-alcohol 52,

5-chloro-7-(2-hydroxy phenyl) quinoline-8-alcohol 56,

5-chloro-7-(4-(dimethylamino) phenyl) quinoline-8-alcohol 60 and

N-[(5-chloro-oxine-7-yl) (2-furyl) methyl] ethanamide 135.

The invention still further relates to and be used for preventing and/or treating illness or its related indication method of being benefited animal from CLK-1 suppresses, this method may further comprise the steps:

A) identify the animal that suffers from the illness of from CLK-1 suppresses, being benefited; And b) gives usefulness compound, its pharmacologically acceptable salt or the prodrug of formula (B) as defined above to described animal.

Advantageously, be used for preventing and/or treating the illness of from CLK-1 suppresses, being benefited or compound, its salt or its prodrug or the optically active isomer of its related indication formula (B) is selected from compound pointed in the table 1 animal.

Even more advantageously be to be used for preventing and/or treating the illness of from CLK-1 suppresses, being benefited or compound, its salt or its prodrug of its related indication formula (B) is selected from animal:

N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] ethanamide 7,

N-[(5-chloro-oxine-7-yl) (2-furyl) methyl] ethanamide 135,

According to preferred version, the illness of being benefited from CLK-1 suppresses is an ischemia/reperfusion injury, inflammation or neurodegenerative disease or dementia.

More preferably, but be not limited to, ischemia/reperfusion injury is the ischemia/reperfusion injury of kidney, heart, cardiac muscle, lung, brain or spinal cord.

According to preferred version, inflammation is a lung inflammation, and the inflammation of liver inflammation or any other organ and any wherein this inflammation are by promoted clinical indication.

According to another embodiment, neurodegenerative disorders is an alzheimer's disease.

Because shown before in mouse that suppressing the CLK-1 activity made that physiology is aging and slow down people such as (, 2005) Liu, this has proved that suppressing the CLK-1 activity in animal can postpone the morbidity and the seriousness of age dependence disease.

In this respect, inventor's compound of disclosing formula of the present invention (B) now also can be effective to treatment or prevention age associated conditions or its related symptoms.Therefore, more preferably, the present invention relates to be used for prevent and/or treat age associated conditions or its related indication method animal, this method may further comprise the steps: a) identify the animal that suffers from the age associated conditions; With

B) to compound, its pharmacologically acceptable salt or the prodrug of described animal to the formula of using (B);

Restricted condition is that the compound of described formula (B) is not one of following compound, its pharmacologically acceptable salt or prodrug:

5-chloro-7-iodine quinoline-8-alcohol 52,

5-chloro-7-(2-hydroxy phenyl) quinoline-8-alcohol 56, or

5-chloro-7-(4-(dimethylamino) phenyl) quinoline-8-alcohol 60.

According to preferred version, above-described method is used to prevent and/or treat and is selected from following age associated conditions or its related symptoms: cardiovascular disorder, and such as atherosclerosis, coronary artery disease and palsy; Peripheral vascular disease; Metabolic disorder, such as type ii diabetes, dyslipidemias mass formed by blood stasis and hypertriglyceridemia; Cancer, such as skin carcinoma, papilloma and age dependency cancer; Ischemia/reperfusion injury is such as kidney, heart, cerebral ischemia and pneumoradiography inductive ephrosis; Inflammation; Neurodegenerative disorders and dementia be alzheimer's disease for example, parkinsonism, Huntington Chorea, dysmnesia and psychosis; Bladder and kidney disorders, such as ephritis, ephrosis, end-stage renal disease (ESRD); Diabetic complication, such as DPN, wound healing difference and retinopathy; Illness in eye, such as related macular degeneration, xeropthalmus, glaucoma, retinitis pigmentosa and cataract; Lung and respiratory disorder are such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF); Flesh and bone disorders be inflammatory arthritis for example, gout and osteoporosis; And skin conditions, such as the cosmetology and the dermatology patient's condition, comprise skin aging, as photoaging and skin carcinoma.

With the animal of formula of the present invention (B) compounds for treating can be the animal of people or any commercially available or domestic value, includes but not limited to ox, horse, pig, cavy, hamster, sheep, rabbit, chicken, dog, cat and bird.More preferably the animal of being treated is the people.

The invention still further relates to pharmaceutical composition, it comprises compound, its pharmacologically acceptable salt or the prodrug of formula (A) as defined above, or any in the prodrug 5,6,136,137,138 and 139 as defined above, itself and one or more nontoxic pharmaceutically acceptable carrier is prepared.

The inventor shows that also formula of the present invention (A) or compound (B) have CLK-1 and suppress active.

Therefore, the invention still further relates to pharmaceutical composition, it comprises compound, its pharmacologically acceptable salt or the prodrug of the formula as defined above (A) or the formula (B) of pharmacy effective dose, to reduce effectively and/or to suppress the CLK-1 activity.

The invention still further relates to pharmaceutical composition, but it comprises that in the prodrug 5,6,136,137,138 and 139 of pharmacy receiving amount any is to reduce effectively and/or to suppress the CLK-1 activity.

According to preferred version, compound of the present invention can have treatment age associated conditions or its related indication active medication combined use with other known, such as for example uniting use with the statins that is used for the treatment of the dyslipidemias mass formed by blood stasis.

Pharmaceutical composition of the present invention can be mixed with solid especially or liquid form is used for oral administration, is used for non-enteron aisle injection, is used for rectal administration or topical.Pharmaceutical composition of the present invention can by in per os, rectum, non-enteron aisle, the pond, intravaginal, intraperitoneal, part (as pulvis, creme, paste or drops), be administered to people and other animal through cheek or as mouth or nasal spray form.These compositions also can comprise auxiliary agent, such as sanitas, and wetting agent, emulsifying agent and dispersion agent.For example parabens, butylene-chlorohydrin, phenol, Sorbic Acid etc. can be guaranteed the effect of prophylaxis of microbial by introducing multiple antibacterium medicine and antifungal drug.Can absorb by the reagent of introducing delay absorption such as the prolongation of aluminum monostearate and gelatinum realization injectable drug form.

In some cases, for the effect of prolong drug, the medicine of wishing to slow down is from subcutaneous and absorption intramuscular injection.This can realize by the liquid suspension that employing has relatively poor water miscible crystallization or an amorphous substance.Therefore the uptake rate of medicine is according to the difference of its dissolution rate and different, and dissolution rate different and different according to crystallographic dimension and crystalline form.As an alternative, absorb can be by with medicine dissolution or be suspended in the oily vehicle and realize in the delay of the medicament forms of parenterai administration.

Be used for peroral administration solid dosage and comprise capsule, tablet, pill, pulvis and granule.In this solid dosage, active compound and the pharmaceutically acceptable vehicle of at least a inert or carrier such as Sodium Citrate or Lin Suanergai and/or weighting agent or extender such as starch, lactose, sucrose, glucose, N.F,USP MANNITOL mix.

Be used for peroral administration liquid dosage form and comprise pharmaceutically useful emulsion, solution, suspension agent, syrup and elixir.Except active compound, liquid dosage form can comprise the normally used inert thinner in this area, such as for example water or other solvents, solubilizing agent and emulsifying agent be ethanol for example, Virahol, ethyl-carbonate, acetate suppresses, benzylalcohol, propylene glycol, glycerine, tetrahydrofuran (THF) alcohol, the fatty acid ester of polyoxyethylene glycol and sorbitanic, and composition thereof.

Compound of the present invention can also carry out administration from the liposome form of phosphatide or the acquisition of other lipid material.Liposome forms by being dispersed in the monolithic in the water-bearing media or the hydration liquid crystallization of splintery.Can use any nontoxic, physiology is acceptable and metabolizable liquid that can form liposome.Preferred lipid is phosphatide and phosphatidylcholine, and the two all is natural or synthetical.

The formulation that is used for the The compounds of this invention topical comprises pulvis, sprays, paste and inhalation.Active compound mixes with pharmaceutically useful carrier and any required sanitas, buffer reagent and the casting charge that can need under aseptic condition.

The actual dose level of the activeconstituents in the pharmaceutical composition of the present invention can change, thereby obtains the active compound of amount that the required treatment of the effective realization of specific patient, composition and administering mode is replied.Selected dosage level will be according to activity, the route of administration of specific compound, treated the seriousness of the patient's condition and different and different by treatment patient's the patient's condition and previous case history.Yet it is in the category well known by persons skilled in the art, begins and increases this dosage gradually up to realizing required curative effect from the compound of the initial dose that is lower than the level that realizes required curative effect.

The present invention will and easily understand with reference to the accompanying drawings according to following examples.These embodiment exemplary illustrations extensive applicability scope of the present invention, and be not intended to limit the scope of the invention.Can change and change and do not break away from the spirit and scope of the present invention.Be used to put into practice the present invention although can use, described preferable methods and material to those method and materials similar or of equal value described herein.

Embodiment

The specificity of embodiment 1:CLK-1 inhibitor

In the eucaryon class, carry out DMQ 9 (DMQ) hydroxylation and in Eubacteria, carry out DMQ 9 (DMQ) hydroxylation by UbiF by CLK-1.Tested the CLK-1 inhibitor molecules in adorned JF496 bacillus coli bacterial strain, this bacterial strain has sudden change and lacks DMQ hydroxylation activity in the ubiF gene.This bacterial strain is supplemented with mouse clk-1 gene or bacillus coli ubiF gene.Should replenish be work and recovered the DMQ hydroxylation.Therefore, the bacterial strain of being rescued can synthesize ubiquinone (Q) rather than gather DMQ.Use two kinds of bacterial strains to estimate for the examination molecule to the synthetic specificity that suppresses of Q through modification.

Material and method

Bacterial strain and substratum

Bacillus coli bacterial strain JF496 (ubiF-) plasmid transfection that comprises mouse clk-1 gene or bacillus coli ubiF gene.For general growth, the vegetative propagation of each bacterial strain ties up under 37 ℃ at 5ml and is supplemented with grow overnight among the LB of Ampicillin Trihydrate (50 μ g/ml).Culture is diluted and measurement OD600 with 1/10.Culture is diluted to OD600 is 0.03 in M9-LB (0.5/0.5:v/v).This mixture oil subsidy is filled with (1mM MgSO ₄, 20 μ M CaCl ₂, 0.5 μ g/mL thiamines, 0.12% acid hydrolysis casein, 40 μ g/mL D-L-methionine(Met), 100 μ g/mL altheines, trace-metal, 0,5% glucose) M9 substratum and LB substratum form.The substratum that comprises Ampicillin Trihydrate (50 μ g/mL) is used for plasmid and selects.Analyze for the HPLC of quinone, JF496-ubiF and JF496-mclk-1 bacterial strain are diluted to from the pre-culture that spends the night to reach OD600 the M9-LB (0.5/0.5:v/v) be 0.03.The bacterium of 750 μ l volumes is transferred in 15 milliliters of test tubes, with bacterial cultures incubation 5 hours under 37 ℃ and jolting.

The extraction of quinone

By the centrifugation harvested cell, with distilled water wash and recentrifuge.Particle is-80 ℃ of following freeze overnight.With cell thawing and be resuspended in 1 milliliter of sodium phosphate buffer (0,1M, pH 7.0), add the Q of 50ng then ₆Extraction contrast as quinone.In order to extract quinone, add 1 milliliter of ethanol and with cell eddy current 30 seconds.Add 1 milliliter of hexane subsequently, then with cell eddy current 2 minutes and carry out centrifugal treating.Phase (hexane) above collecting, following phase are extracted once more and are carried out centrifugal treating.Phase above collecting then also joins it in previous extract.With about 1 hour of top phase lyophilize, and particle remained under-80 ℃.Particle is resuspended in the moving phase of 300 μ l, carries out HPLC then and analyze.

The HPLC method

Use has photodiode array detector and Beckman UltrasphereODSS (the Beckman System Gold HPLC analytic sample of post of 4.6mm * 25cm).Use 32Karat software (Beckman) that data are analyzed.Use methyl alcohol-ethanol (beginning is 70% methyl alcohol-30% ethanol, after 6 minutes, is 30% methyl alcohol-70% ethanol), carry out the moving phase wash-out with the flow velocity of 1 ml/min and reach 20 minutes and separate quinone.Under 275 nanometers, detect the peak of DMQ 9 (DMQ) and ubiquinone (Q).

Result and short discussion about the drug specificity test

The JF496 bacterium of expression mouse CLK-1 and bacterium DMQ-hydroxylase UbiF is handled (Fig. 1) and handles (Fig. 2) with compound 52 with compound 7.Use HPLC to analyze by the quinone distribution that these processing obtain.As if

compound

7 and 52 can suppress the CLK-1DMQ hydroxylase activity, and this obtains the support of gathering of DMQ precursor.In contrast to this, the UbiF activity is not subjected to the attack of any compound, because DMQ does not gather in processed cell.This disparate impact has characterized the specificity that compares the CLK-1 effect with UbiF, even these two kinds of enzymes transmit identical functions: the DMQ hydroxylation.Observed effect to CLK-1 is not because the non-specific active institute of some of compound causes in bacillus coli because CLK-1 and UbiF the two all under identical bacterium genetic background, expressed.

Embodiment 2: measure CLK-1 and suppress

In order to confirm that being confirmed as is that the male compound is the real inhibitor of CLK-1 in bacteria screening, the inventor adopts and tests the quinone content of measuring in the mammalian cell based on the secondary of HPLC.CLK-1 is demethoxy quinone (DMQ) hydroxylase, the penultimate stride in its catalysis Q biosynthesizing.The inhibition of CLK-1 causes gathering of reacting precursor DMQ.This experimental measurement the endogenous cell level of Q and precursor DMQ thereof.In the whole cells for examination, DMQ exists level normally inappreciable, therefore the strongly inhibited of CLK-1 is caused the deep change of the HPLC quinone curve of processed cell.

Material and method

Will be from bacteria screening the fresh dilution of compounds identified be dissolved in the dimethyl sulfoxide (DMSO) (DMSO) with the concentration of 10mM and spend the night.The dilution of compound (in DMSO) is added in the hole of RAW264.7 cell, and this cell is the cell system of the mouse macrophage of Abelson murine leukemia virus conversion.In the day before yesterday of compound treatment, cell bed board (1 * 105 cell in every hole) DulbeccoShi modification Eagle's medium (being supplemented with 10% foetal calf serum, 1% penicillin/streptomycin, 100 μ M Sodium.alpha.-ketopropionates), and is placed moistening CO ₂Insulation can (5%CO ₂, 37 ℃) in spend the night.After adding compound, with plate at moistening CO ₂(5%CO ₂, 37 ℃) and descended incubation 24 hours.

After 24 hours, sucking-off substratum from the hole; Cell washs with PBS, adds 500 μ l RIPA damping fluids (15 minutes, room temperature) and jolting leniently then in every hole.Afterwards, sample is moved in 1.5 milliliters of Eppendorf tubes.The HPLC level ethanol that adds 500 μ l in each pipe adds the HPLC level hexane of 500 μ l then, then with the violent eddy current of centrifuge tube 1 minute, centrifugal then (3000 * g, 15 minutes, room temperature).After centrifugal, visible two tangible layers in each pipe.The upper strata is moved in the new Eppendorf tube carefully, cover, carry out lyophilize then with the needle-like oilhole.Behind sample drying, in each pipe, add the 70%HPLC level methyl alcohol of 320 μ l: 30% ethanol (v/v).To manage violent eddy current then 20 seconds, centrifugal 10 seconds (14000xg), then with sample transfer in the HPLC bottle that is covered.Then each sample being carried out HPLC analyzes.By making sample flow cross 250mm C18 ODS post, use ethanol: the methyl alcohol gradient, and use photodiode array detector under 275 nanometers, to detect quinone and analyze.Use the stratographic visual control to determine the activity of each compound of interest of a plurality of dosage.

Table 1 has shown through measuring as the compound tabulation with active CLK-1 inhibitor.

CLK-1 suppresses in mammalian cell result and short discussion

Fig. 3 has shown the typical HPLC curve that obtains from the quinone extract of the sample of RAW 264.7 mouse macrophages.The quinone that last figure representative is extracted from undressed scavenger cell, wherein significantly essential substance is ubiquinone-9 or Q.When handling cell with the CLK-1 inhibitor, observe second quinine peak of appearance, it represents gathering of Q precursor.The cell that this curve representation is handled with compound 135.Therefore, can easily discern the CLK-1 inhibitor by with the naked eye clear with the HPLC curve of analyzing the cell of handling with the candidate inhibitor of supposition simply.Detect the clone of various human and mouse, between them, except endogenous ubiquinone level difference, do not had main difference.

This means that the unique conceivable mode that can detect Q loss in the cell is the inhibition of CLK-1.This second trial is therefore exceedingly useful and very powerful aspect the ability of its rapid evaluation CLK-1 inhibitor.Fig. 4-8 has shown the HPLC curve with the compound 7,23,69,47 of different concns or 53 mouse macrophages of handling.Observing increases the effect of compound concentration to the distribution of DMQ9 in the processed cell and Qg.

Embodiment 3: measure the effect that CLK-1 suppresses

The CLK-1 activity can influence cell ROS level by the cell content of regulating Q, and Q generates ROS by electron transport chain and ROS removing contribution.Therefore, the inhibition of expection CLK-1 will cause the change of cell ROS level.In addition, be DNA in the cell as one of macromole of the target of ROS, cause dna damage.Can expect that dna damage also can be owing to CLK-1 suppresses to be lowered.Therefore the inventor uses two tests to measure these factors in the viable cell, and is as follows.

Material and method

The ROS test

Use DCF-DA (2,7-dichlorofluorescein diacetate esters) to estimate the ROS level of cell.Lipophilic DCF-DA betransported by cytolemma and arrives tenuigenin, and be enzymatically converted to by the tenuigenin esterase hydrophilic 2,7-dichlorofluorescein (DCFH).The ROS of cell is oxidized to DCF with DCFH, and it is fluorescigenic molecule.In order to estimate the CLK-1 inhibition, at first, will inoculate (every hole 1 * 10 to the effect that ROS generates ⁵Individual) mouse macrophage (RAW264.7) in 96 orifice plates handled 24 hours with the CLK-1 inhibitor.Then with the DCF-DA of cell and 20 μ M in Hanks balanced salt solution (HBSS) 37 ℃ of following incubations 30 minutes.Use fluorescence to read plate device (excitation wavelength 488 nanometers and emission wavelength 520 nanometers) then and monitor the fluorescence that derives from cell DCF.Using commercially available BCA albumen test kit to measure for the proteinic amount in the prospect hole.ROS water-glass with cell is shown the proteic average DCF of the fluorescence/mg that derives from 3 repeated experiments subsequently.

The COMET test

Under the condition of existence/shortage CLK-1 inhibition compound, in 12 orifice plates, inoculate (every hole 2.5 * 10 ⁶Individual cell) RAW264.7 cell.After 24 hours, cell 1mM H ₂O ₂Handled 1 hour, to induce the dna damage of ROS mediation.With cell washed twice in ice-cold PBS, scrape then among the ice-cold PBS of 575 μ l then.This mixture of 75 μ l is taken out and joins among the ice-cold PBS of 750 μ l.Take out 10 μ l aliquots and mix with the fused low melting-point agarose of 90 μ l.This cell-agarose mixture of 75 μ l is moved out on the slide glass with transfer pipet, and slide glass lies against 4 ℃ of following also lucifuges and reaches 15 minutes.

Afterwards, slide glass is immersed in advance in the refrigerative lysate and at 4 ℃ placed 1 hour lucifuge down.Then, take out slide glass and after removing excessive lysate, it is immersed in the freshly prepd basic solution (pH 13).At room temperature placed slide glass then 1 hour, lucifuge.

Remove excessive basic solution, slide glass at room temperature is immersed in the 1XTris-Borate/EDTA damping fluid (TBE) reaches 5 minutes then.Lucifuge places slide glass the electrophoresis apparatus that is full of 1x TBE and applied voltage 20 minutes once more.Taking-up slide glass and lucifuge are immersed in and reach 5 minutes in 70% ethanol after 20 minutes, form thin layer 37 ℃ of following dry airs up to agarose on slide glass then.

Behind these 37 ℃ of incubations, in the dark the SYBR of the dilution of adding 50 μ l is green in each exsiccant agarose dish reaches 10 minutes, washes the accent dyestuff in the distilled water by being immersed in then.Blot excessive water, then slide glass is in the dark carried out dry air once more.Behind complete drying, under fluorescent microscope, observe slide glass and detect the existence that showed cell is examined the DNA afterbody (comet) of trace.Data are represented with the percentage ratio of the cell that comprises comet usually, have been counted 10 visuals field.

The result and the short discussion of the effect of CLK-1 inhibitor pair cell ROS level

At the quinone curve, cell ROS level and dna damage aspect show prototype CLK-1 inhibitor be compound 7 and 135 three figure as shown in Figure 9.These molecules are found and reduce H in the scavenger cell ₂O ₂To inducing of ROS, the effectiveness of compound 7 stronger (figure B).These molecules in scavenger cell to by H ₂O ₂The inductive dna damage has suitable antagonism effect (figure C), and compound 7 is extremely effective.These data have emphasized that the inventor has reduced the cell levels of ROS and the damage that is produced subsequently.

Embodiment 4: the CLK-1 inhibitor can be used for treating the ischemia reperfusion injury of kidney in rat.

Following research evaluation the administration of CLK-1 inhibitor in the renal ischaemia model, be used to prevent and/or reduce the practicality of reperfusion injury.

Brief description and experimental technique

(9 to 11 weeks the are big) male Sprague-Dawley rat of growing up is anaesthetized and is carried out the right side nephrectomy by the center line laparotomy.Reach the temporary ischemic of realizing left renal artery in 60 minutes by blocking left renal artery and vein.Beginning once a day intraperitoneal (Lp.) before surgical operation on the 3rd day gives with 15 running days all groups is treated (vacation: no ischemic, accept salt solution (0.9%NaCI), be used for experiment first: ischemic adds vehicle, and treatment: ischemic adds

compound

7 or 52).The treatment winding be subjected to 2.5mg/kg/ days Lp. (1mg/ml) compound 7 or the compound 52 of 20mg/kg/ days (4mg/ml).The the 0th, 3,7,11 and 14 day by measure creatinine and the blood urea nitrogen (BUN) in the serum and urinate in creatinine and protein content evaluate renal function.Use formula CrCI=(uCr*uV)/(sCr x 1440x weight) to calculate creatinine clearance (ml/min/100g).Use the Coomassi method to measure the urine protein concentration in 24 hours in the urine sample.Analyze histo pathological change (hematoxylin and the eosin of nephridial tissue by optical microscopy; H﹠amp; The painted slide glass of E), observe the necrosis of blood vessel epithelial cell, vasodilation, the tubular and marrow hyperemia of protein according to the suggestion of Racusen (2).Use one-way analysis of variance when pointing out, to use Turkey post-hoc test that the data of collecting are carried out statistical study then.The result of p＜0.05 is considered to significance on statistics.

Ischemia reperfusion injury result's brief description

Renal function: serum creatinine

Initial results shows that serum creatinine level did not have the statistics difference in every group at the 0th day.In with compound 7 or the group handled, significantly be lower than at 3-14 days serum creatinines and be used to the group (Figure 10) of testing first.

Renal function: blood urea nitrogen (BUN)

Initial results shows that every group does not have the statistics difference at the 0th day BUN.In the groups of handling with

compound

7 and 52, at the 3rd, 7,11 and 14 day, BUN significantly was lower than and is used to the group (Figure 11) of testing first.

Renal function: CrCl

In every group, the CrCl at the 0th day does not have the statistics difference.In the group of handling with compound 7 or compound 52, be significantly higher than at the 3rd and 7 day CrCl and be used to the group of testing first.In the groups of handling with

compound

7 or 52, at the 11st and 14 day, CrCl did not have the statistics difference with the group that is used to first test, yet, be used to the group of testing (Figure 12) first in the 14th day apartment.

Renal function: urine protein

In every group, at the 0th day, twenty-four-hour urine albumen did not have the statistics difference.In the groups of handling with

compound

7 or 52, at the 3rd day, twenty-four-hour urine albumen significantly was lower than and is used to the group of testing first.In with

compound

7 or 52 groups of handling, at the 7th and 4 day, twenty-four-hour urine albumen is compared with the group that is used to first test did not have the statistics difference.In the groups of handling with

compound

7 and 52, at the 11st day, twenty-four-hour urine albumen significantly was lower than and is used to the group (P=0.033) of testing first (Figure 13).

The CLK-1 inhibitor is to the concise and to the point discussion of the effect of renal ischaemia-reperfusion injury

Renal function data (serum creatinine.Blood urea nitrogen (BUN).CrCl and urine protein) show that again the beginning of ischemic event first three day uses compound 7 (2.5mg/kg) and compound 52 (20mg/kg) treatment and lasting 11 running days once a day, with compare with the control group that is used to first to test, a kidney rat model behind 60 minutes ischemics effectively prevents renal ischaemia/reperfusion injury with similar degree.In all cases, the renal function in the false group is unaffected.Behind ischemia/reperfusion the 3rd and 7 day, to the protection particular significant effect of some parameters of renal function.

Embodiment 5:CLK-1 inhibitor is to the effect of lipopolysaccharide-induced acute lung injury.

Following research evaluation the application in the relevant rat model in the body of the acute pneumonia disease of bringing out of CLK-1 inhibitor by the lipopolysaccharides that is administered systemically (LPS).

The brief description of experimental technique

The male Sprague-Dawley rat of heavy 225 to 250 grams (n=5 to 10/group) is handled to induce injury of lung with lipopolysaccharides (LPS).(i.p.) accepts compound 7 or as the N-ethanoyl-L-halfcystine (NAC) with reference to thing in the rat peritoneum that heals with medicine.Compound in the vehicle that comprises DMA/PG/Tween 80 and water by the i.p. administration.Gave in one day and use dose, continue 3 running days, last dosed administration is contacting preceding 1 hour of LPS.Drug media thing shown in control rats is accepted.Freshly prepd medicine with 5 or the volume of 10ml/kg or 5ml/kg give and use.Mouse is put to death by the excessive use urethane in after i.p.LPS or salt solution administration 24 hours, and collects blood alveolar fluid (BALF) and be used for total cell count and difference cell counting, and LPO and TNF-α measure and the protein content protein content.

LPS inductive injury of lung result's brief description and discussion

Be presented at LPS (6mg/kg, Lp.) three days before once a day Lp. give drug compound 7 (5mg/kg or 10mg/kg) to deriving from total cell count and neutrophilic granulocyte counting, protein and the TNF-alpha content that is exposed to the rat under saline (contrast) or the LPS, the effect of LPO level is shown in Figure 14-17 among the BALF.N-acetylcysteine (NAC; 0.5g/kg, Lp., LPS first three day once a day) as with reference to compound by administration.Speak briefly, compound 7 reduces the level that can compare with the eye-level in the rat for the treatment of with NAC that is increased to of total cell of BALF and neutrophil effectively.This chemical combination is when the BALF total cell count not being produced any effect for the time spent separately.Similarly, compound 7 reduce effectively LPO among the BALF be increased to can with in the level that compare with the eye-level in the rat of NAC treatment.Compound 7 does not reduce the proteinic increase of BALF.Compound 7 (10mg/kg) is effective to significantly reduce the BALFTNF-alpha levels.In addition, the histologic analysis (not shown) has shown 5mg/kg, and the compound 7 of Lp is effective to reduce the intensity (by alveolus wall thinner thickness in the lung for the treatment of) and the inflammatory response (number of inflammatory cell is less) of gap figure.

Generally speaking, these experiments have shown that compound 7 reduces the application of BALF total cell count and neutrophilic granulocyte counting, lipid hydroperoxide and histology tuberculosis change (not shown) in the rat of attacking with LPS.The reduction of TNF-alpha levels also is observed when the compound 7 of i.p. dosed administration 10mg/kg.

Because neutrophilic granulocyte flows into and LPO is subjected to the known markers of inflammation thing that the CLK-1 inhibitor is protected, novel quinoline is used for the application that this inflammation is able to promoted clinical indication and has represented useful especially embodiment.

Embodiment 6: treat the brain amyloid-beta 1-40 of hAPP751SL transgenic mice and the effect of amyloid-beta 1-42 level with compound 138.

Animal: initial day male hAPPSL transgenosis (tg) mouse (C57BL/6background) in research reaches 5 monthly ages (± 2 week).Transgenosis hAPP751SL animal constitutive character external table intelligent APP751 has London (V717I) and Swedish (K670M/N671L) sudden change under the regulation and control of neuronal tissue's specific murine-Thy-1 promotor.Because the London sudden change, amyloid-beta 1-42 still mainly occurs in cortex and the hippocampus with high level expression in whole brain.High-caliber amyloid-beta 1-42 with since morning of 3 months under many age the amyloid patch in CNS form relevant.The enhancing of observed brain A β 1-42 level forms relevant with early patch in this mouse model.A β 1-42 promotes the amyloid deposition and promotes the formation of dense deposit; Although perhaps A β 1-40 has reverse effect.Therefore, the generation that influences this mortality amyloid peptide can be favourable, particularly about the possible treatment aspect to patients with Alzheimer disease.

Treat: compound 138 is formulated in the carboxymethyl cellulose (CMC) and respectively is administered to animal with one day twice (maximum dose level) oral (p.o.) once a day, reach 60 running days.Therapeutic dose is respectively 5,20,50 and 2 * 50mg/kg b.w./sky, or uses vehicle (CMC).

Tissue preparation: flow to end up to whole blood with pouring into physiology (0.9%) salt solution in the transgenic mice heart; Promptly take out brain then and right hemisphere dipping was fixed in the 4% fresh Paraformaldehyde 96 1 hour.Then, hemisphere is transferred in 15% sucrose solution be used for cryprotection.Second day, with brain freezing and preservation under-80 ℃ on dry ice.With left brain hemisphere on dry ice at once quick freezing being determined at 4 amyloid-beta 1-40 and amyloid-beta 1-42 in the brain homogenate fraction,

Measure: use the brain hemisphere (comprising that olfactory bulb does not still have cerebellum) of every animal to be used for estimating brain A β 1-40 and A β 1-42 in 4 brain fractions (it is TBS, Triton X-100 and FA).A β 1-40 and A β 1-42 use the highly sensitive ELISA test kit (TK40HS by the The GENETICS Company manufacturing of Switzerland ^TMTK42HS ^TM).

The result:

The level of solubility A β 1-40 and A β 1-42

The water-soluble A β 1-40 and the A β 1-42 fraction of TBS extract fraction security personnel cerebral tissue, although in Triton x-100 fraction, similar fibriilar littler polymkeric substance is dissolved.Formic acid (FA) fraction comprises insoluble polymerization A β.The result is shown in Figure 18-23.Compound 138 significantly reduces TBS (5 and 20mg/kg; Figure 19) and Triton X-100 fraction (5,20 and 50mg/k; A β 1-42 level Figure 21).In TBS fraction (Figure 18 and 19), but not in Triton fraction (Figure 20 and 21), similar but inapparent effect can be observed in measuring A β 1-40, wherein all one data point very denses in the group of two compounds 138.

In the FA fraction, the average A β 1-42 value of all dosage all is lower than the value of vehicle contrast, thus 5 and 20mg/kg concentration under effect be significant (Figure 23).

Therefore, the generation of 138 pairs of APP processing of compound and A β peptide has unusual effect, although this effect is more remarkable to A β 1-42.Significantly A β 1-42 reduction effect all is observed in multiple brain fraction, and it is up to standard the different polymerization state of amyloid beta (monomer, oligopolymer, protofibril).

Conclusion:

In brain, the tangible A amyloid beta 1-42 that can be observed compound 138 reduces effect.The most surprising and result uniqueness are that compound 138 can see through brain fraction (monomer, oligopolymer, protofibril) to reduce A β 1-42 level.Result's importance is emphasized that by this model A β 1-42 level is high, even compares such as the Tg2576 mouse with other transgenic models.Therefore, even in this aggressive model, the A β 1-42 level that compound 138 reduces in the whole brain fraction.Although the effect about A β 1-42 is the clearest, compound 138 also reduces the A β 1-40 in the brain fraction to a certain extent.The more significant effect of 138 couples of toxicity A of compound β 1-42 peptide causes the ratio of A β 1-42/A β 1-40 to reduce, and this has the treatment benefit.

Embodiment 7: from order A synthetic compound.

Compound in order A can make by the strategy that adopts the following stated.Group except 5-Cl as F or Me, is allowed in this chemistry equally.

Embodiment 7.1:N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] ethanamide (compound 7) { method A_1}

5-chloro-oxine (1.5g, 8.7mmol), 3,4-dimethoxy benzaldehyde (2.85g, 17.4mmol) and ethanamide (1.03g, 17.4mmol) intimate mixture be sealed in the microwave reaction container, container placed microwave reactor (Biotage Initiator) and be heated to 180 ℃ reach 1 hour, refrigerative reaction mixture and ethyl acetate are ground, leach the solid of gained.This solid further grinds with ethanol, obtains title compound, is white solid (2.2g, 65%).MS387(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：10.28(bs，1H)，8.93-8.97(m，1H)，8.74(d，1H)，8.47(d，1H)，7.69-7.75(m，2H)，6.91-6.94(m，1H)，6.87(d，1H)，6.74(d，1H)，6.62(d，1H)，3.70(s，3H)，3.71(s，3H)，1.94(s，3H)。

Embodiment 7.2:N-((5-chloro-oxine-7-yl) (quinoline-3-yl) methyl) ethanamide (compound 19) { method A_1}

5-chloro-oxine (0.25g, 1.4mmol), quinoline-3-formaldehyde (0.44g, 2.8mmol) and ethanamide (0.165g, 2.8mmol) intimate mixture be sealed in the microwave reaction container, this container placed microwave reactor (Biotage Initiator) and be heated to 180 ℃ reach 0.5 hour, the solid of gained is ground and leached to refrigerative reaction mixture and ethyl acetate, this solid further grinds with ethanol and obtains title compound, be white solid (0.236g, 44%).MS?377(M ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：10.52(bs，1H)，8.96-9.01(m，2H)，8.85(s，1H)，8.51(d，1H)，8.11(s，1H)，7.95-8.01(dd，2H)，7.71-7.79(m，4H)，7.58(t，1H)，6.87(d，1H)，2.01(s，3H)。

Embodiment 7.3:N-((5-chloro-oxine-7-yl) (pyridin-3-yl) methyl) ethanamide (compound 32) { method A_1}

5-chloro-oxine (0.25g, 1.4mmol), 3-pyridylaldehyde (0.299g, 2.8mmol) and ethanamide (0.165g, 2.8mmol) intimate mixture be sealed in the microwave reaction container, this container placed microwave reactor (Biotage Initiator) and be heated to 180 ℃ reach 0.5 hour, the solid of gained is ground and leached to refrigerative reaction mixture and ethyl acetate, this solid further grinds with ethanol and obtains title compound, be white solid (0.123g, 27%).MS?327(M ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：10.49(bs，1H)，8.97(s，1H)，8.90(d，1H)，8.47-8.51(m，3H)，7.72-7.77(m，2H)，7.64(d，1H)17.33-7.36(dd，1H)，6.70(d，1H)，1.97(s，3H)。

Embodiment 8: from sequence B 1 synthetic compound

Compound in sequence B 1 can adopt strategy described below to make.It is that the synthetic that adopted by the sequence A compound is replenished and is particularly suitable for wherein R ₁It is the compound of alkyl or aryl.Group except 5-C1 as F or Me, is allowed in this chemistry equally.

Method B1_1: intermediate 2-((5-chloro-oxine-7-yl) methyl)-1H-isoindole-1,3 (2H)-diketone synthetic

With 2-(hydroxymethyl)-1H-isoindole-1,3 (2H)-diketone (14.8g, 83mmol) be added drop-wise to abundant stirring and be cooled to 0 ℃ 5-chloro-oxine (15g, 83mmol) in the solution in the vitriol oil (150ml), behind reinforced the finishing, reaction is heated to 100 ℃ spends the night, make the mixture room temperature of rising again, be poured into then on the trash ice (1000ml), filter the yellow mercury oxide of gained, wash also vacuum-drying with water and spend the night, this solid and ethanol and hexane grind, obtain pale solid (16.7g, 59%).MS?338(M ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：10.24(bs，1H)，8.95(d，1H)，8.48(d，1H)，7.41-7.92(m，4H)，7.72(dd，1H)，7.57(s，1H)，4.96(s，2H)。

Method B1_2: intermediate 2-((8-(benzyl oxygen base)-5-chloroquinoline-7-yl) methyl)-1H-isoindole-1,3 (2H)-diketone synthetic

With 2-((5-chloro-oxine-7-yl) methyl)-1H-isoindole-1,3 (2H)-diketone (16.68g, 49mmol), bromobenzyl (12.63g, 73.5mmol) and salt of wormwood (13.61g, 98mmol) dissolve/be suspended in the dimethyl formamide (250ml) and be heated to 60 ℃ and reach 1.5 hours, be poured into reaction in the cold water (1000ml) and filter pale precipitation thing, water thorough washing.This solid vacuum-drying obtains pure compound (20.8g, 98%).MS?446([M+H ₂O] ⁺)； ¹HNMR(DMSO-d ₆)，400MHzδ：9.09(d，1H)，8.55(d，1H)，7.83-7.91(m，4H)，7.75(dd，1H)，7.64(s，1H)，7.55-7.61(m，2H)，7.347.44(m，3H)，5.53(s，2H)，4.96(s，2H)。

Method B1_3: intermediate 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl) methylamine synthetic

With a hydrazine hydrate (2.28ml, 46.6mmol) join 2-((8-(benzyl oxygen base)-5-chloroquinoline-7-yl) methyl)-1H-isoindole-1,3 (2H)-diketone (2.0g, 4.7mmol) in the suspension in methyl alcohol (50ml), mixture was refluxed 1 hour, then solvent removed in vacuo, with resistates and hot ethanol (～10ml) grind and heat filtering, concentrated ethanol obtains light brown solid (1.1g, 79%), and it need not any further purifying and can be used for subsequent reaction.MS?299(MH ⁺)； ¹HNMR(CDCl ₃)，400MHz?δ：9.02(d，1H)，8.54(d，1H)，7.59(s，1H)，7.51(dd，1H)，7.41-7.44(m，2H)，7.31-7.39(m，3H)，5.50(s，2H)，3.87(s，2H)。

Method B1_4: intermediate 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl)-N-(phenylbenzene methylene radical) methylamine synthetic

With 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl) methylamine (407mg, 1.36mmol) and benzophenone imine (247mg, 1.36mmol) mixture together 60 ℃ of down heating, after 1 hour, the solid and the methyl alcohol of gained are ground and filters, obtain title compound, be light brown solid (480mg, 76%).MS?463(M ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：9.06(d，1H)，8.57(d，1H)，7.96(s，1H)，7.73(dd，1H)，7.54-7.62(m，5H)，7.39-7.48(m，3H)，7.20-7.31(m，7H)，5.29(s，2H)，4.61(s，2H)。

Method B1_5: intermediate 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl)-N-(phenylbenzene methylene radical) ethamine synthetic

With methyl iodide (147mg, 1.03mmol) and 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl)-N-(phenylbenzene methylene radical) methylamine (480mg, 1.03mmol) be dissolved in the anhydrous tetrahydro furan (0.5ml) and be cooled to 0 ℃, in this mixture, drip potassium tert.-butoxide (128mg, 1.16mmol) solution in tetrahydrofuran (THF) (0.13ml), with mixture rise again room temperature and after 1 hour the concentration response thing.Be dissolved in the ethyl acetate resistates and water and salt water washing,, obtain title compound, be white solid (480mg, 97%) with anhydrous magnesium sulfate drying organic extraction and vacuum concentration.MS?477(M ⁺)； ¹H?NMR(CDCl ₃)，400MHzδ：8.89(d，1H)，8.47(d，1H)，8.15(s，1H)，7.60(d，2H)，7.42(dd，1H)，7.15-7.38(m，10H)，7.03-7.09(m，3H)，5.20(d，1H)，5.18(q，1H)，4.81(d，1H)，1.23(d，3H))。

Method B1_6: intermediate 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl) ethamine synthetic

To 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl)-N-(phenylbenzene methylene radical) ethamine (550mg, 1.15mmol) add oxammonium hydrochloride (144mg in the solution in methyl alcohol (5ml), 2.07mmol) and mixture at room temperature stirred spend the night, the concentration response thing and with residuum with hydrochloric acid (5ml, 5% aqueous solution) handle, and with ethyl acetate extraction to remove benzophenone, water layer is with sodium hydroxide solution (5M) alkalization and with ethyl acetate extraction (3 * 5ml), the organic extraction salt water washing that merges, use anhydrous magnesium sulfate drying, filter and concentrate, obtain title compound (230mg, 64%).MS313(M ⁺)； ¹H?NMR(CDCl ₃)，400MHzδ：9.00(d，1H)18.49(d，1H)，7.68(s，1H)，7.51(dd，1H)，7.31-7.44(m，5H)，5.50(q，2H)，4.59(q，1H)，1.46(bs，2H)，1.26(d，3H))。

Method B1_7: intermediate N (1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl) ethyl) ethanamide synthetic

To with ice-cooled 1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl) ethamine (220mg, 0.7mmol) and triethylamine (0.12ml, 0.84mmol) dripping acetyl chloride (66mg in the solution of stirring in methylene dichloride (2.5ml), 0.84mmol) solution in methylene dichloride (0.5ml), reactant at room temperature stirs and spends the night, use methylene dichloride (5ml) dilution then, mixture is used saturated sodium carbonate solution, water and salt water washing continuously, and use anhydrous magnesium sulfate drying, solvent removed in vacuo, obtain title compound (240mg, 96%).MS?354(M ⁺)； ¹H?NMR(CDCl ₃)，400MHzδ：8.90(d，1H)，8.44(d，1H)，7.54(d，2H)，7.40-7.45(m，2H)，7.26-7.37(m，3H)，6.22(bd，1H)，5.61(d，1H)，5.46(d，1H)，5.36(q，1H)，1.77(s，3H)，1.36(d，3H)。

Synthesizing of embodiment 8.1:N-(1-(5-chloro-oxine-7-yl) ethyl) ethanamide (compound 47)

To ice-cooled N-(1-(8-(benzyl oxygen base)-5-chloroquinoline-7-yl) ethyl) ethanamide (0.7g, 1.97mmol) under nitrogen atmosphere, add boron trichloride (1M in the solution of stirring in methylene dichloride (20ml), in methylene dichloride, 4ml, 4mmol) solution in methylene dichloride (5ml).Mixture at room temperature stirred 3 hours then, then reactant is poured into carefully with also using dichloromethane extraction in the ice-cooled water (100ml), the organic extraction salt water washing that merges, use anhydrous magnesium sulfate drying, filter and concentrate, thick material obtains title compound by silica gel chromatography purifying (0-10%MeOH:100-90%EtOAc), be white solid (0.45g, 86%).MS265(M ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：10.17(bs，1H)，8.95(d，1H)，8.47(d，1H)，8.39(d，1H)，7.70(dd，1H)，7.64(s，1H)，5.41(q，1H)，1.88(s，3H)，1.35(d，3H)。

Embodiment 9: from sequence B 2 synthetic compounds

Compound in the sequence B 2 can make by either party's method described below:

Method B2_1

Method B2_2

Embodiment 9.1:N-(oxine-2-yl) pyridine-2-carboxamide (compound 69) { method B2_2}

To the chlorinated picoline hydrochloride (89mg that is under 0 ℃, 0.5mmol) add triethylamine (0.42ml in the suspension in tetrahydrofuran (THF), 3mmol) with 2-amino-oxine (160mg, 1.0mmol), with rise again room temperature and stir and to spend the night of mixture, solvent removed in vacuo, thick material is by the HPLC purifying, obtain required product (15mg, 11%).MS?265(M ⁺)； ¹HNMR(DMSO-d ₆)，400MHzδ：10.79(bs，1H)，9.70(bs，1H)，8.81(d，1H)，8.53(d，1H)，8.43(d，1H)，8.28(d，1H)，8.16(t，1H)，7.75-7.80(m，1H)，7.36-7.41(m，2H)，7.08(d，1H)。

Embodiment 9.2:N-(oxine-2-yl)-2-pyridine-2-yl acetamide (compound 70) { method B2_1}

With 4-pyridyl acetic acid hydrochloride (87mg, 0.5mmol) be dissolved in the dimethyl formamide (4ml), in this solution, add hydroxybenzotriazole (68mg, 0.5mmol), N-(3-dimethylaminopropyl)-N '-ethyl-carbodiimide hydrochloride (96mg, 0.5mmol) and triethylamine (0.28ml, 2.0mmol), mixture was at room temperature stirred 30 minutes, and adding 2-amino-oxine in reaction (80mg, 0.5mmol) also stirring is spent the night, solvent removed in vacuo, be dissolved in resistates in the methylene dichloride (2ml) and wash with water, the vacuum concentration organic phase, thick material is by the HPLC purifying, obtain required product (11mg, 8%).MS?280(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ：11.00(bs，1H)，9.48(bs，1H)，8.52(d，2H)，8.27(d，1H)，8.21(d，1H)，7.39(d，2H)，7.28-7.34(m，2H)，7.07(d，1H)，3.88(s，2H).(10mg，7％)。MS280(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHz?δ10.92(bs，1H)，9.35-9.55(bs，1H)，8.53(d，1H)，8.17-8.29(m，2H)，7.78(t，1H)，7.44(d，1H)，7.26-7.35(m，3H)，7.08(d，1H)，4.04(bs，2H)。

Embodiment 9.3:2-(3-hydroxy phenyl)-N-(oxine-2-yl) ethanamide (compound 71) { method B2_1}

Prepare title compound similarly according to method B21 described in the above embodiment 9.2.Yield=12mg (9%).MS?295(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHz?δ10.84(bs，1H)，9.43(bs，1H)，9.34(bs，1H)，8.21-8.28(dd，2H)，7.27-7.35(m，2H)，7.11(t，1H)，7.07(d，1H)，6.81(s，1H)，6.79(d，1H)，6.64(d，1H)，3.70(s，2H)。

Embodiment 10: from sequence B 3 synthetic compounds

Compound in the sequence B 3 can adopt scheme as described below to make:

Method B3_1: intermediate 5-chloro-7-iodo-8-isopropoxy quinoline synthetic

To enteroquinol (18g, 58.9mmol) in the suspension of dimethyl sulfoxide (DMSO), drip salt of wormwood (32.57g, 235.6mmol) and 2-N-PROPYLE BROMIDE (10.87g, 88.4mmol), mixture at room temperature stirred to spend the night then it is poured in the saturated ammonium chloride solution, this mixture dichloromethane extraction (3 * 300ml), the organic extraction that merges is successively with sodium hydroxide (2M), water and salt water washing, and use anhydrous magnesium sulfate drying, solvent removed in vacuo is also used ethyl acetate: the mixture of hexane carries out the silica gel chromatography wash-out, obtain required compound (11.0g, 55%).MS347(M ⁺)； ¹H?NMR(CDCl ₃)，400MHzδ：8.92(d，1H)，8.50(d，1H)，7.97(s，1H)，7.51(dd，1H)，5.38(sept，1H)，1.42(d，6H)。

Synthesizing of method B3_2: intermediate: 3-(5-chloro-8-isopropoxy quinoline-7-yl) phenol

With 5-chloro-7-iodo-8-isopropoxy quinoline (500mg, 1.7mmol), (3-hydroxy phenyl) boric acid (238mg, 1.7mmol) and PdPCy3 (18mg, 0.028mmol) dissolve/be suspended in acetonitrile/water and (be respectively 4ml: in mixture 1ml), and adding sodium carbonate solution (2M, 5ml), then mixture being heated to 80 ℃ under nitrogen also stirred 1 hour simultaneously, TLC shows that initial sulfonation compound exhausts fully, and solvent removed in vacuo is dissolved resistates/be suspended in the ethyl acetate, water and salt water washing, organic phase obtains pure substantially product (TLC and NMR) (350mg, 77%) with anhydrous magnesium sulfate drying and vacuum concentration.MS?314(MH ⁺)； ¹H?NMR(CDCl ₃)，400MHz?δ：9.01(bs，1H)，8.92(d，1H)，8.51(d，1H)，7.55(s，1H)，7.47(dd，1H)，7.35(t，1H)，7.32(s，1H)，6.97-7.02(m，2H)，4.36(sept，1H)，1.12(d，6H)。

Embodiment 10.1:5-chloro-7-(3-hydroxy phenyl) quinoline-8-alcohol (compound 57) { method B3_3}

With 3-(5-chloro-8-isopropoxy quinoline-7-yl) phenol (475mg, 1.52mmol) be dissolved in and be cooled to-78 ℃ in the methylene dichloride (4ml) and under nitrogen, the mixture that drips the boron trichloride dimethyl thioether is at methylene dichloride (2M, 3.04ml, 6.08mmol) in solution, and mixture stirred 2 hours at-78 ℃, add methyl alcohol (20ml) carefully and with the reactant room temperature of rising again, solvent removed in vacuo also adds other a methyl alcohol (20ml), vacuum concentration once more, this process repeats twice again to obtain crude product, and it obtains title compound (315mg, 76%) by the HPLC purifying.MS?272(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ9.5-10.5(vbs，1H)，9.02(d，1H)，8.56(d，1H)，7.78(dd，1H)，7.72(s，1H)，7.29(t，1H)，7.17(bs，1H)，7.11(d，1H)，6.80(d，1H)。

Method B3_2: intermediate 5-chloro-8-isopropoxy-7-(2-methoxypyridine-3-yl) quinoline synthetic

Similarly prepare title compound according to aforesaid method B3_2.MS?329(MH ⁺)； ¹HNMR(CDCl ₃)，400MHzδ8.96(d，1H)，8.54(d，1H)，8.21(d，1H)，7.77(d，1H)，7.63(s，1H)，7.48-7.53(dd，1H)，6.99-7.02(dd，1H)，4.57(sept，1H)，3.97(s，3H)，1.06(d，6H)。

Embodiment 10.2:5-chloro-7-(2-methoxypyridine-3-yl) quinoline-8-alcohol (compound 61.) { method B3_3}

Similarly prepare title compound according to the method B3_3 in the foregoing description 10.1 from 5-chloro-8-isopropoxy-7-(2-methoxypyridine-3-yl) quinoline.MS?288(MH ⁺)； ¹HNMR(DMSO-d ₆)，400MHzδ10.15(bs，1H)，9.00(d，1H)，8.54(d，1H)，8.23(d，1H)，7.76-7.81(m，2H)，7.64(s，1H)，7.12(dd，1H)，3.86(s，3H)。

Method B3_2: intermediate 4-(5-chloro-8-isopropoxy quinoline-7-yl)-N, accelerine synthetic

Similarly prepare title compound according to aforesaid method B3_2.MS?341(MH ⁺)； ¹HNMR(CDCl ₃)，400MHzδ8.95(d，1H)，8.49(d，1H)，7.69(s，1H)，7.63(d，2H)，7.43-7.46(dd，1H)，6.80(d，2H)，4.51(sept，1H)，3.02(s，6H)，1.11(d，6H)。

Embodiment 10.3:5-chloro-7-(4-(dimethylamino) phenyl) quinoline-8-alcohol (compound 60) { method B3_3}

From 4-(5-chloro-8-isopropoxy quinoline-7-yl)-N, accelerine similarly prepares title compound according to the method B3_3 in the foregoing description 10.1.MS?300(MH ⁺)； ¹HNMR(DMSO-d ₆)，400MHzδ9.02(d，1H)，8.57(d，1H)，7.77-7.86(m，4H)，7.55-7.68(bs，2H)，3.13(s，6H)。

Embodiment 11: from sequence B 4 synthetic compounds

Compound in the sequence B 4 can adopt scheme as described below to make:

Method B4_1

Embodiment 11.1:2-(((2-p-methoxy-phenyl) amino) methyl) quinoline-8-alcohol (compound 77) { method B4_1}

To oxine-2-formaldehyde (87mg, 0.5mmol) 1, add 2-anisidine (62mg in the solution of 2-ethylene dichloride (2ml), 0.5mmol), and with mixture stirring 30 minutes, add triethoxy sodium borohydride (95mg then, 45mmol), reactant is at room temperature stirred spend the night then, then evaporated in vacuo, be dissolved in resistates in the methylene dichloride and wash with water, water extracts with methylene dichloride is counter, and the organic extraction of merging obtains crude product through super-dry and vacuum concentration, it is by HPLC purifying (74mg, 53%).MS?281(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ9.51(bs，1H)，8.22(d，1H)，7.52(d，1H)，7.32-7.43(q，2H)，7.09(d，1H)，6.83(d，1H)，6.67(t，1H)，6.48-6.56(dd，2H)，5.96(t，1H)，4.59(d，2H)，3.84(s，3H)。

Embodiment 11.2:2-(((3-p-methoxy-phenyl) amino) methyl) quinoline-8-alcohol (compound 75) { method B4_1}

Similarly prepare title compound according to the method B41 in the foregoing description 11.1.Yield=71mg (51%).MS?281(MH ⁺)； ¹H?NMR(DMSO-d ₆)，400MHzδ9.74(bs，1H)，8.27(d，1H)，7.53(d，1H)，7.36-7.45(q，2H)，7.11(d，1H)，7.08(t，1H)，6.70(t，1H)，6.36(d，1H)，6.32(s，1H)，6.16(d，1H)，4.54(d，2H)，3.69(s，3H)。

Embodiment 12: phosphoric acid ester prodrug synthetic

Phosphoric acid ester prodrug (compound 5)

Scheme of discussing in this section and rules can be applied to other the compound in this sequence equally.

Synthetic route

a)NaHCO ₃，n-Bu ₄NHSO ₄，H ₂O-CH ₂Cl ₂，0℃～RT.；

b)K ₂CO ₃，DMF，25℃；

C) 1,10%Pd/C, EtOH,

Benzyl chloride methyl phosphorodithioate A's is synthetic

With phosphate dibenzyl ester (2.64g, 9.48mmol), sodium bicarbonate (3.18mL, 11.97mmol) and 4-n-butyl ammonium hydrogen sulfate (3.2g 9.48mmol) is dissolved in the water (80ml), add methylene dichloride (DCM, 90mL), mixture 0 ℃ of vigorous stirring 10 minutes, is added in the chloro sulfuric acid chloromethyl ester (1.17mL among the DCM (15mL) then, 11.37mmol) simultaneously at room temperature continuously vigorous stirring spend the night, separate organic layer, use the salt water washing, dry (MgSO ₄) and evaporation, resistates by using the sudden strain of a muscle silica gel chromatography of ethyl acetate/hexane (1: 1) as elutriant, is obtained the pure substance of 1.39g (44.8%).MS?327(MH ⁺)；H?NMR(CDCl ₃)，400MHz?δ5.09(d，2H，J＝8Hz)，5.62(d，2H，J＝16Hz)，7.41-7.28(m，10Hz)。

Synthesizing of prodrug derivatives:

Synthesizing of compound 5:

Rules: with compound 7 (0.5g, 1.29mmol), potash solid (0.36g, 2.58mmol) be suspended in anhydrous N, dinethylformamide (DMF, 5mL), add the benzyl chloride methyl phosphorodithioate (A that is dissolved among the DMF (2mL) then, 0.42g, 1.29mmol), and under nitrogen atmosphere 0 ℃ of stirring, reaction mixture was at room temperature stirred under nitrogen atmosphere 16 hours, reaction mixture is poured in the water (50mL), with EtOAc extract (3 * 25mL), the salt water washing of the extract of merging, drying (MgSO ₄) and evaporate to dryness.LC-MS hour oily resistates of thick resistates is 97% pure under 254nm.Therefore need not carry out purifying in addition this moment, this oily resistates can be used in the later step, and MS 677 (MH+) and isotopic pattern meet; ¹HNMR (DMSO), 400MHz δ 1.99 (s, 3H), 3.64 (s, 3H), 3.68 (s, 3H), 4.72 (d, 1H, J=4Hz), 4.74 (d, 1H, J=3.5Hz), 4.89 (d, 2H1J=7.8Hz), 5.96-6.05 (m, 2H), 6.47 (d, 1H, J=8.08Hz), 6.58 (d, 1H, J=8.33Hz), 6.64 (dd, 1H, J=8.58﹠amp; 2.02Hz), 6.80 (d, 1H, J=2.02Hz), 7.10-7.23 (m, 10H), 7.43 (dd, 1H, J=8.58﹠amp; 8.58Hz), 7.54 (s, 1H), 7.55 (d, 1H, J=8.33Hz), 8.45 (dd, 1H, J=6.82﹠amp; 1.76Hz), 8.80 (dd, 1H1J=4.04﹠amp; 1.76Hz).

Synthesizing of compound 5:

Rules: under nitrogen atmosphere, in the suspension of 10%Pd/C in EtOH, drip the solution of B in EtOH (3mL), drip cyclohexadiene (0.25mL then; 10eq, 2.58mmol), reaction mixture was at room temperature stirred 12 hours, on diatomite, leach catalyzer, and with filtrate evaporation almost extremely dried (still keeping～10% EtOH after the evaporation), then thick resistates and anhydrous diethyl ether are ground, leach precipitated solid, and use ether rinse, obtain the pure substance of 0.065g (60%).MS 497 (MH+) and isotopic pattern meet; ¹H NMR (DMSO), 400MHz δ 1.52 (s, 3H), 3.3 (s, 3H), 3.49 (s, 3H), 5.63 (m, 2H), 6.40 (d, 1H, J=9.8Hz), 6.61 (m, 2H), 6.88 (s, 1H), 7.46 (m, 2H), 8.29 (d, 1H, J=8.58Hz), 8.74 (d, 1H, J=3.2Hz), 8.83 (d, 1H, 7.7Hz).

Phosphoric acid ester prodrug (compound 138)

Step 1:P (OBut) 2 (=O) (OCH2Cl) I's is synthetic

Synthesize I according to following route:

Step 2:138's is synthetic

I (8.8g) and compound 7 (as synthetic among the embodiment 7.1) reaction in dimethyl formamide (DMF) under the condition that K2CO3 exists; form ((7-((acetylamino) (3; the 4-Dimethoxyphenyl) methyl)-and 5-chloroquinoline-8-yl) the oxygen base) methyl di-t-butyl phosphoric acid ester II (9.6g; the HPLC yield is 96%); II (5.0g) reacts in the mixture of EtOAc/Et2O with HCl then; form 138, yield 85%.

Table 1

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Claims

1. the compound of formula (A), its pharmacologically acceptable salt or prodrug,

Perhaps R ₁For-CH (R ₃) NR ₄R ₅, wherein

■ R ₄=H or C ₁-C ₄Alkyl;

■ R ₅=H or

■ R ₃Be selected from: H, methyl, phenyl, benzyl, the 2-chloro-phenyl-, 2-p-methoxy-phenyl, 4-chloro-phenyl-, 4-aminomethyl phenyl, the 4-isopropyl phenyl, 3-hydroxy phenyl, 4-hydroxy phenyl, the 4-p-methoxy-phenyl, 4-cyano-phenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, the 2,4 dichloro benzene base, 3,4-Dimethoxyphenyl, 4-methoxycarbonyl phenyl, 3-methoxycarbonyl phenyl, N-methyl-benzamide base, N-(3-methoxy-propyl) benzoylamino, N, N-dimethyl benzamide base, 4-(morpholine-4-base carbonyl) phenyl, 4-(pyridine-2-yl) phenyl, 4-(1H-pyrazol-1-yl) phenyl, pyridine-2-base, pyridin-3-yl, pyridin-4-yl, 6-(morpholine-4-yl) pyridin-3-yl, the 2-furyl, 3-furyl, 2-(morpholine-4-yl) pyridin-3-yl, 2-methoxypyridine-3-base, 4-methoxypyridine-3-base, 2-thienyl, 2-butyl-1H-imidazol-4 yl, quinoline-3-base, quinolyl-4, oxine-2-base, 1H-indol-3-yl, 1H-indoles-4-base and 1H-indoles-7-base;

Perhaps R ₁For

Restricted condition is that the compound of described formula (A) is not one of compound pointed in the annex 1;

The compound of described formula (A) is the form of one of its optically active isomer or the form of its mixture when comprising at least one asymmetric center.

2. the compound of claim 1, wherein R ₄Be H or methyl, and R ₆Be selected from methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, phenyl, benzyl, dimethylaminomethyl, phenylethyl, cyclohexyl, diphenyl methyl, pyridine-2-base and pyridin-3-yl.

3. the compound of claim 2 is selected from following:

N-((3, the 4-Dimethoxyphenyl) (oxine-7-yl) methyl) ethanamide 2,

N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl) benzamide 3,

7-(amino (3, the 4-Dimethoxyphenyl) methyl)-5-chloroquinoline-8-alcohol 13,

N-(1-(5-chloro-oxine-7-yl)-2-phenylethyl) ethanamide 14,

N-((2-butyl-1H-imidazol-4 yl) (5-chloro-oxine-7-yl) methyl) ethanamide 15,

N-((5-chloro-oxine-7-yl) (2-methoxypyridine-3-yl) methyl) ethanamide 16,

N-((5-chloro-oxine-7-yl) (quinolyl-4) methyl) ethanamide 17,

N-((5-chloro-oxine-7-yl) (quinoline-3-yl) methyl) ethanamide 19,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl) methyl benzoic acid ester 21,

3-((acetylamino) (5-chloro-oxine-7-yl) methyl) methyl benzoic acid ester 22,

N-((5-chloro-oxine-7-yl) (4-cyano-phenyl) methyl) ethanamide 23,

N-((5-chloro-oxine-7-yl) (4-pyridine-2-base phenyl) methyl) ethanamide 24,

N-((5-chloro-oxine-7-yl) (4-(1H-pyrazol-1-yl) phenyl) methyl) ethanamide 25,

N-((5-chloro-oxine-7-yl) (4-hydroxy phenyl) methyl) ethanamide 26,

N-((5-chloro-oxine-7-yl) (3-hydroxy phenyl) methyl) ethanamide 27,

N-((5-chloro-oxine-7-yl) (6-methoxypyridine-3-yl) methyl) ethanamide 28,

N-((5-chloro-oxine-7-yl) (pyridine-2-yl) methyl) ethanamide 29,

N-((5-chloro-oxine-7-yl) (pyridin-4-yl) methyl) ethanamide 30,

N-((5-chloro-oxine-7-yl) (pyridin-4-yl) methyl) acetamide hydrochloride 31,

N-((5-chloro-oxine-7-yl) (pyridin-3-yl) methyl) ethanamide 32,

N-((5-chloro-oxine-7-yl) (pyridin-3-yl) methyl) acetamide hydrochloride 33,

N-((5-chloro-oxine-7-yl) (oxine-2-yl) methyl) ethanamide 34,

N-((5-chloro-oxine-7-yl) (1H-indol-3-yl) methyl) ethanamide 36,

N-((5-chloro-oxine-7-yl) (1H-indoles-4-yl) methyl) ethanamide 37,

N-((5-chloro-oxine-7-yl) (1H-indoles-7-yl) methyl) ethanamide 38,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl)-N, N-dimethyl-benzamide 39,

4-((acetylamino) (5-chloro-oxine-7-yl) methyl)-N-methyl-benzamide 45,

N-(1-(5-chloro-oxine-7-yl) ethyl) ethanamide 47,

N-((oxine-7-yl) methyl) ethanamide 48,

N-(2-furyl methyl)-oxine-7-base methane amide 53,

N-(2-thienyl methyl)-oxine-7-base methane amide 54,

N-(2-methoxy-benzyl)-oxine-7-base methane amide 55,

5-chloro-7-(3-hydroxy phenyl) quinoline-8-alcohol 57,

5-chloro-7-(4-hydroxy phenyl) quinoline-8-alcohol 58,

N-(2-(5-chloro-oxine-7-yl) phenyl) ethanamide 59,

5-chloro-7-(2-methoxypyridine-3-yl) quinoline-8-alcohol 61,

5-chloro-7-pyridin-4-yl quinoline-8-alcohol hydrochloride 62,

N-(3, the 4-Dimethoxyphenyl)-oxine-7-base methane amide 63,

N-(3-p-methoxy-phenyl)-oxine-7-base methane amide 64,

N-(3-methoxy-benzyl)-oxine-7-base methane amide 65,

N-(3, the 4-dimethoxy-benzyl)-oxine-7-base methane amide 66,

N-(2-p-methoxy-phenyl)-oxine-7-base methane amide 67,

N-(pyridin-3-yl methyl)-oxine-7-base methane amide 68,

N-(oxine-2-yl) pyridine-2-base methane amide 69,

N-(oxine-2-yl)-2-pyridine-2-yl acetamide 70,

2-(3-hydroxy phenyl)-N-(oxine-2-yl) ethanamide 71,

2-(4-hydroxy phenyl)-N-(oxine-2-yl) ethanamide 72,

N-(oxine-2-yl)-2-(2-thienyl) ethanamide 73,

2-(((3-methoxy-benzyl) amino) methyl) quinoline-8-alcohol 74,

2-(((3-p-methoxy-phenyl) amino) methyl) quinoline-8-alcohol 75,

2-(((2-thienyl methyl) amino) methyl) quinoline-8-alcohol 76,

2-(((2-p-methoxy-phenyl) amino) methyl) quinoline-8-alcohol 77,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] ethanamide 78,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl] ethanamide 79,

N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl] ethanamide 80,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl] butyramide 81,

N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl] butyramide 82,

The N-[(2-chloro-phenyl-) (oxine-7-yl) methyl]-3-Phenylpropionamide 93,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl]-3-methylbutyryl amine 94,

N-[(8-hydroxyquinoline-7-yl) (phenyl) methyl]-3-methylbutyryl amine 110,

N-[(3, the 4-Dimethoxyphenyl) (oxine-7-yl) methyl]-3-methylbutyryl amine 112,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl]-3-methylbutyryl amine 113,

The N-[(4-chloro-phenyl-) (oxine-7-yl) methyl] cyclohexane carboxamide 121,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] propionic acid amide 122,

N-[(3, the 4-Dimethoxyphenyl) (oxine-7-yl) methyl]-3-Phenylpropionamide 124,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] valeramide 125,

N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl] valeramide 126,

N-[[4-(diethylamino) phenyl] (oxine-7-yl) methyl] valeramide 127,

N-[(2, the 4-dichlorophenyl) (oxine-7-yl) methyl] butyramide 129,

N-[(8-hydroxyquinoline-7-yl) (2-thienyl) methyl] cyclohexane carboxamide 131.

4. be selected from following prodrug:

5. be used for suppressing the active method of CLK-1 at cell, this method may further comprise the steps:

A) provide and wherein need to suppress the active cell of CLK-1,

B) make the compound of this cells contacting formula (B):

Perhaps R ₁For-CH (R ₃) NR ₄R ₅, wherein

■ R ₄=H or C ₁-C ₄Alkyl;

■ R ₅=H or

Perhaps R ₁For

C) the CLK-1 activity in the mensuration cell.

6. the method for claim 5, the compound of wherein said formula (B) is selected from compound pointed in the table 1.

7. the method for claim 6, the compound of wherein said formula (B) is selected from:

N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] ethanamide 7

5-chloro-7-iodine quinoline-8-alcohol 52,

5-chloro-7-(2-hydroxy phenyl) quinoline-8-alcohol 56,

5-chloro-7-(4-(dimethylamino) phenyl) quinoline-8-alcohol 60 and

N-[(5-chloro-oxine-7-yl) (2-furyl) methyl] ethanamide 135.

8. be used for preventing and/or treating illness or its related indication method of being benefited animal from CLK-1 suppresses, this method may further comprise the steps:

A) identify the animal that suffers from the illness of from CLK-1 suppresses, being benefited; With

B) to compound, its pharmacologically acceptable salt or the prodrug of described animal to the formula of using (B),

Perhaps R ₁For-CH (R ₃) NR ₄R ₅, wherein

■ R ₄=H or C ₁-C ₄Alkyl;

■ R ₅=H or

Perhaps R ₁For

The compound of formula (B) is the form of one of its enantiomorph or the form of its mixture when comprising at least one asymmetric center.

9. the method for claim 8, the compound of wherein said formula (B) is selected from compound pointed in the table 1.

10. the method for claim 8, the compound of wherein said formula (B) is selected from:

N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] ethanamide 7,

N-[(5-chloro-oxine-7-yl) (2-furyl) methyl] ethanamide 135,

11. the method for claim 8, wherein said illness are ischemia/reperfusion injury or inflammation.

12. be used for preventing and/or treating age associated conditions or its related indication method animal, this method may further comprise the steps:

A) identify the animal that suffers from the age associated conditions; With

B) give compound, its pharmacologically acceptable salt or the prodrug of using the formula (B) in the claim 8 to described animal;

5-chloro-7-iodine quinoline-8-alcohol 52,

5-chloro-7-(2-hydroxy phenyl) quinoline-8-alcohol 56, or

5-chloro-7-(4-(dimethylamino) phenyl) quinoline-8-alcohol 60.

13. the method for claim 12, wherein said age associated conditions is selected from: cardiovascular disorder, peripheral vascular disease, metabolic disorder, cancer, neurodegenerative disorders, dementia, bladder and kidney disorders, diabetic complication, illness in eye, lung and respiratory disorder, flesh and bone disorders and skin conditions.

14. the method for claim 13, wherein said neurodegenerative disorders is an alzheimer's disease.

15. the method for claim 11, wherein said ischemia/reperfusion injury are the ischemia/reperfusion injuries of kidney, heart, cardiac muscle, lung, brain or spinal cord.

16. the method for claim 11, wherein said inflammation are the inflammation of lung inflammation or any other organ.

17. the method for claim 8, wherein said animal is the people.

18. the method for claim 12, wherein said animal is the people.

19. pharmaceutical composition, it comprises compound, its pharmacologically acceptable salt or the prodrug of the formula (A) of claim 1, and at least a pharmaceutically acceptable carrier.

20. pharmaceutical composition, it comprises compound, its pharmacologically acceptable salt or the prodrug of the formula (A) of claim 2, and at least a pharmaceutically acceptable carrier.

21. pharmaceutical composition, it comprises compound, its pharmacologically acceptable salt or the prodrug of the formula (A) of claim 3, and at least a pharmaceutically acceptable carrier.

22. pharmaceutical composition, it comprises prodrug and at least a pharmaceutically acceptable carrier of claim 4.

23. pharmaceutical composition, it compound, its pharmacologically acceptable salt or prodrug of formula (A) that comprises the claim 1 of pharmacy effective dose is to reduce effectively and/or to suppress the CLK-1 activity.

24. pharmaceutical composition, it compound, its pharmacologically acceptable salt or prodrug of formula (B) that comprises the claim 8 of pharmacy effective dose is to reduce effectively and/or to suppress the CLK-8 activity.

25. the pharmaceutical composition of claim 22, it prodrug that comprises pharmacy effective dose is to reduce effectively and/or to suppress the CLK-1 activity.

Annex 1

Compound number The chemical name of compound ??1 N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl]-the 2-methyl propanamide ??4 N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] valeramide ??7 N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] ethanamide ??9 N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] butyramide ??10 N-[(5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl] propionic acid amide ??20 N-((5-chloro-oxine-7-yl) (3-furyl) methyl) ethanamide ??42 N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl)-2-phenoxy-acetamide ??43 N-((5-chloro-oxine-7-yl) (3, the 4-Dimethoxyphenyl) methyl) niacinamide ??49 5,7-dichloroquinoline-8-alcohol ??50 5,7-two bromoquinolines-8-alcohol ??51 5,7-dijodoxichinoline-8-alcohol ??52 5-chloro-7-iodine quinoline-8-alcohol ??56 5-chloro-7-(2-hydroxy phenyl) quinoline-8-alcohol ??60 5-chloro-7-(4-(dimethylamino) phenyl) quinoline-8-alcohol ??83 N-[(5-chloro-oxine-7-yl) (4-aminomethyl phenyl) methyl] ethanamide ??84 N-[(5-chloro-oxine-7-yl) (4-chloro-phenyl-) methyl] ethanamide ??85 N-[(5-chloro-oxine-7-yl) (2-chloro-phenyl-) methyl] ethanamide ??86 N-[(5-chloro-oxine-7-yl) (2,4 dichloro benzene base) methyl] ethanamide ??87 N-[(5-chloro-oxine-7-yl) (2-thienyl) methyl] ethanamide ??88 N-[(5-chloro-oxine-7-yl) (4-isopropyl phenyl) methyl] ethanamide ??89 N-[(5-chloro-oxine-7-yl) (2-furyl) methyl] propionic acid amide ??90 N-[(5-chloro-oxine-7-yl) (2-p-methoxy-phenyl) methyl] propionic acid amide ??91 N-[(5-chloro-oxine-7-yl) (4-chloro-phenyl-) methyl] propionic acid amide ??95 N-[(5-chloro-oxine-7-yl) (phenyl) methyl] butyramide ??96 N-[(5-chloro-oxine-7-yl) (phenyl) methyl] valeramide

????97 N-[(5-chloro-oxine-7-yl) (2-furyl) methyl]-the 2-methyl propanamide ????98 N-[(5-chloro-oxine-7-yl) (2-p-methoxy-phenyl) methyl] butyramide ????99 N-[(5-chloro-oxine-7-yl) (2-p-methoxy-phenyl) methyl]-the 2-methyl propanamide ????100 N-[(5-chloro-oxine-7-yl) (4-p-methoxy-phenyl) methyl] butyramide ????101 N-[(5-chloro-oxine-7-yl) (4-p-methoxy-phenyl) methyl] valeramide ????102 N-[(5-chloro-oxine-7-yl) (4-aminomethyl phenyl) methyl] butyramide ????103 N-[(5-chloro-oxine-7-yl) (4-aminomethyl phenyl) methyl] valeramide ????104 N-[(5-chloro-oxine-7-yl) (4-aminomethyl phenyl) methyl]-the 2-methyl propanamide ????105 N-[(5-chloro-oxine-7-yl) (4-chloro-phenyl-) methyl] butyramide ????106 N-[(5-chloro-oxine-7-yl) (4-chloro-phenyl-) methyl] valeramide ????107 N-[(5-chloro-oxine-7-yl) (2-chloro-phenyl-) methyl] valeramide ????108 N-[(5-chloro-oxine-7-yl) (2-thienyl) methyl]-the 3-Phenylpropionamide ????109 N-[(5-chloro-oxine-7-yl) (4-isopropyl phenyl) methyl]-the 2-methyl propanamide ????114 N-[(8-hydroxyquinoline-7-yl) (4-isopropyl phenyl) methyl]-3-methylbutyryl amine ????115 N-{ (5-chloro-oxine-7-yl) [4-(dimethylamino) phenyl] methyl } butyramide ????116 N-[(5-chloro-oxine-7-yl) (2-thienyl) methyl] butyramide ????117 N-[(5-chloro-oxine-7-yl) (2-thienyl) methyl] valeramide ????118 N-[(5-chloro-oxine-7-yl) (2-thienyl) methyl]-the 2-methyl propanamide ????123 N-[(5-chloro-oxine-7-yl) (2,4 dichloro benzene base) methyl] propionic acid amide ????132 N-[(5-chloro-oxine-7-yl) (phenyl) methyl] ethanamide ????133 N-[(5-chloro-oxine-7-yl) (2-p-methoxy-phenyl) methyl] ethanamide ????134 N-[(5-chloro-oxine-7-yl) (2-p-methoxy-phenyl) methyl] valeramide ????135 N-[(5-chloro-oxine-7-yl) (2-furyl) methyl] ethanamide