CN101495454A - Novel PDE4 inhibitors - Google Patents

Abstract

PDE4 inhibitors, pharmaceutical compositions including the inhibitors, and methods of treatment using the inhibitors are disclosed. The compounds are illustrated by Formulas 1-9 in the specification. The compounds can be used to treat a variety of disorders mediated by phosphodiesterase, and TNF-a, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, arthritis, respiratory distress syndrome, allergic rhinitis, neurogenic inflammation, pain, rheumatoid arthritis, central nervous system disorders, cardiovascular disorders, and tumor progression.

Description

Novel PDE 4 inhibitors

Invention field

The present invention is intended to inhibitors of phosphodiesterase-4, and it can be specific or non-specific for various phosphodiesterase-4 subfamilies.

Background of invention

Several enzyme inhibitorss based on cyclic nucleotide phosphodiesterase (PDE) have been studied and have been used for the treatment of various human diseasess.Among them, the PDE4 inhibitor has shown the potentiality at treatment asthma and chronic obstructive pulmonary disease, though they are often with side effect.Described side effect is considered to that other PDE families or PDE4 subfamily produce owing to having suppressed.

The concentration of cyclic nucleotide phosphodiesterase (PDE) control " second messenger " cyclic amp and cyclic guanosine monophosphate (cAMP and cGMP).PDEs is hydrolyzed into 5 with cAMP and cGMP '-AMP and 5 '-GMP.Be presented among Fig. 1 to the hydrolysis of AMP by the catalytic cAMP of PDE.PDE is hydrolysis cGMP in an identical manner.These second messengers mediate the biological response of a series of hormone and neurotransmitter, and for example smooth muscle contraction, glycogenolysis, apoptosis and growth control of metabolic process.Have 11 PDE families, they are subdivided into again surpasses 60 kinds of isotypes.Each family has different substrate specificities and has selective depressant.PDE 4,7 and 8 selective hydrolysis cAMP, and PDE 5,6 and 9 is that cGMP is specific.PDE 1,2,3,10 and 11 these enzymes demonstrate the activity for these two kinds of Nucleotide.The homology of PDEs is classified as the terminal montage of three zone: N-zones, adjusted and controlled territory and near the catalyst structure domain of C-stub area.The function of N-stub area is unknown, and adjusted and controlled territory comprises various structure divisions and be assumed in regulation and control PDEs the catalytic activity aspect play a role.The catalyst structure domain of all 11 PDE families is shared about 30 to 50% amino acid identity.The conservative property of catalyst structure domain has indicated in these families, and identical inhibitor also can be shared by these families.In fact, reported, a kind of selectivity PDE5 inhibitor, viagra has the IC of 3.5nM ₅₀, and it also demonstrates the inhibition for PDE4, has the IC of 8 μ M ₅₀Several non-selective PDE inhibitor have also been identified.

Be studied with the avtive spot bonded PDE4 inhibitor of described enzyme and be used for the treatment of asthma and chronic obstructive pulmonary disease.Since disadvantageous side effect, for example vomiting, and nearest concern concentrates on the PDE4 inhibitor that only suppresses one of subfamily PDE4A, B, C or D.PDE4D is present in the central nervous system, and PDE4B is present in (people such as Wang, Mol.Pharmacol.56,170-174 (1999)) in neutrophilic granulocyte and the monocyte.Nearest discovery shows that PDE4D is ishemic stroke gene (people such as Gretarsdottir, Nature Genet.35:131-138 (2003)).This hint PDE4D may be relevant with vomiting.Current research concentrates on and develops optionally is the PDE4 inhibitor of target with one of described 4 kinds of PDE4 subfamilies.

Current novel cpd and the composition that still needs therapeutic ground to suppress PDE4 (ideally so that minimum mode is reduced in side effect).The invention provides such compound and composition.

Summary of the invention

The present invention is intended to specific and nonspecific PDE4 inhibitor, comprises the pharmaceutical composition of described inhibitor, the method for synthetic described inhibitor and use the methods of treatment of described inhibitor.

Described inhibitor is the benzene of replacement or the six-membered Hetero-aromatic of replacement, and wherein said hetero-aromatic ring comprises one or two theheterocyclic nitrogen atom.Described ring replaces with ether, thioether or amine groups, and wherein the alkyl on described ether, thioether or amine is a haloalkyl, for example methyl fluoride, difluoromethyl or trifluoromethyl.Described ether, thioether or amine groups for 5 or 6 yuan of ring cycloalkyl or heterocyclic radical part speeches be in the ortho position and for alkyl, ketone group or amido (C (=O)-NHR) be in contraposition for partly.

In one embodiment, described haloalkyl is a difluoromethyl.In another embodiment, described cycloalkyl is a cyclopentyl, and perhaps described heterocyclic group is tetrahydrofuran base or pyrrolidyl.In the 3rd embodiment, the described ketone group that is in contraposition for described alkyl partly is C _3-5Moieties, for example C ₄Moieties comprises normal-butyl, sec-butyl, isobutyl-and tertiary butyl part.Described compound can be included in the pharmaceutical composition, and described pharmaceutical composition comprises the described compound and the pharmaceutically acceptable carrier of significant quantity.

Described compound can be used for the treatment of the Mammals people for example who suffers from various illnesss, described illness comprises for example asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), the eosinophilic granuloma, optimum or neoplasm dermatoses with other, endotoxin shock (and relevant symptom, for example in the laminitis and the angina of Malaysia and China), septic shock, ulcerative colitis, Crohn's disease, the reperfusion injury of cardiac muscle and brain, inflammatory arthritis (inflammatoryarthritis), osteoporosis, chronic glomerulonephritis, atopic dermatitis, urticaria, adult respiratory distress syndrome, infant respiratory distress syndrome, chronic obstructive pulmonary disease in the animal, diabetes insipidus, allergic rhinitis, anaphylaxis conjunctivitis, vernal conjunctivitis, arterial restenosis, atherosclerosis, neurogenic inflammation, pain, cough, rheumatoid arthritis, ankylosing spondylitis, transplant rejection and graft versus host disease (GVH disease), gastroxia, bacterium, the Sepsis of fungi or virus induction or septic shock, inflammation and cytokine mediated chronic tissue's sex change, osteoarthritis, cancer, emaciation, amyotrophy (muscle wasting), dysthymia disorders, dysmnesia, unipolar depression (monopolar depression), acute and chronic neurodegenerative disorders (neurodegenerative disorderswith inflammatory components) with inflammatory component, Parkinson's disease, alzheimer's disease, spinal cord injuries receptor, craniocerebral injury, multiple sclerosis, the cancerous invasion of tumor growth and healthy tissues (cancerous invasion of normal tissues).By the compound as herein described of using significant quantity or the precursor compound that forms described compound in vivo, can treat these illnesss and prevent these illnesss in some cases.

The accompanying drawing summary

Fig. 1 is such figure, and it has shown the hydrolysis by catalytic cAMP of PDE and cGMP.

Fig. 2 is such figure, and it has shown the structural domain structure of 11 PDE families.

Fig. 3 is such chart, it has shown compound 1 and 9 and the relative reactivity of rolipram in TNF-α suppresses, be shown as %LPS to μ M concentration, wherein use square data presented representation compound 1, represent rolipram with trilateral data presented representation compound 9 with circular data presented.

Detailed Description Of The Invention

By with reference to hereinafter detailed description and with reference to give a definition, will understand better the present invention.

As used herein; " alkyl "; and with other groups of prefix " alkane (alk) "; such as alkoxyl (alkoxy), alkanoyl (alkanoyl), alkenyl (alkenyl), alkynyl (alkynyl) etc., refer to can for linearity or branch or its combination carbochain. The example of alkyl comprises methyl, ethyl, propyl group, isopropyl, butyl, the second month in a season and the tert-butyl group, amyl group, hexyl etc. Alkyl can replace with the hydrogen-bonded group of promotion with the PDE4 acceptor, for example OH, SH and NH₂Group. " alkenyl ", " alkynyl " term similar with other comprise the carbochain that contains at least one unsaturated C--C key.

Term " aryl " refers to aromatic substituents, and it is monocycle or condenses together a plurality of rings. When being formed by a plurality of rings, at least one ring in the described makeup ring is aromatic. Preferred aryl substituent is phenyl.

As used herein, term " is used altogether " and is intended to comprise and uses simultaneously compound described herein and at least treatment of the second form, and they can be formulated in the single formulation together; Basically use simultaneously, use in turn, or each other with certain remarkable time interval continuous administration.

Term " compound " is intended to comprise compound described herein and their pharmacy acceptable salts.

Term " cycloalkyl " is meant and does not contain heteroatomic carbocyclic ring, comprises single, double and the trinucleated saturated carbon ring, and the fused rings system.Thereby such fused rings system can comprise for example phenyl ring formation fused rings system of a partially or completely undersaturated ring, for example benzo-fused carbocyclic ring (benzofused carbocycles).Cycloalkyl also comprises the fused rings system such as spiro-condensed ring (spirofused ring) system.The example of cycloalkyl comprises cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, naphthane, diamantane, indanyl, indenyl, fluorenyl, 1,2,3,4-tetralin, or the like.Similarly, " cycloalkenyl group " is meant the carbocyclic ring that does not contain heteroatoms but contain the two keys of at least one non-aromatic C--C, comprises single, double and trinucleated fractional saturation carbocyclic ring, and benzo-fused cycloolefin.The example of cycloalkenyl group comprises cyclohexenyl, indenyl, or the like.

Unless specify that in addition term " is mixed " and comprised one or more O, S or N atom.For example, Heterocyclylalkyl and heteroaryl comprise such loop systems, and described loop systems contains at least one and is no more than two O, S and/or N atom in ring, comprise the mixing of this type of atom.Described heteroatoms displaced loop carbon atom.Therefore, for example, heterocycle C ₅Alkyl is the five-ring that contains 2 to 4 carbon atoms.The example of heteroaryl comprises the six-ring with one or two theheterocyclic nitrogen atom, for example pyridyl, pyridazinyl, pyrimidyl and pyrazinyl, and five-ring heterocycle (comprise with phenyl ring condensed those), for example furyl, benzofuryl, dibenzofuran group, thienyl, benzothienyl, pyrryl, indyl, pyrazolyl, indazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, benzimidazolyl-, oxadiazole base and thiadiazolyl group.

The example of Heterocyclylalkyl comprises azetidinyl, pyrrolidyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuran base, imidazolinyl, pyrrolidin-2-one, piperidines-2-ketone and thio-morpholinyl, and wherein tetrahydrofuran base and pyrrolidyl are particularly preferred.

Unless specify in addition, term " amine " comprises primary, the second month in a season and tertiary amine, although primary amine is can be preferred, because they will help combining of promotion and associated receptor.

Term " halogen " comprises fluorine, chlorine, bromine and iodine atom.

Term " Mammals " comprises people and other animals, for example dog, cat, horse, pig and ox.Use compound described herein, can just relevant illness treat other animals except the mankind with human sufferer.Term " animal " only is used for pointing out the mankind for other members of animal kingdom.Described compound has the treatment applicability, and is to be used for the treatment of Mammals (generally speaking), particularly human.

Term " replaces " alternatively, comprises replacement and unsubstituted part.Therefore, for example, the aryl of Qu Daiing can be represented pentafluorophenyl group or benzyl ring alternatively.In addition, a plurality of parts of Qu Daiing alternatively, for example alkaryl is meant that alkyl and aryl are replaced alternatively.If in a plurality of parts only a part replaced alternatively, then will be specifically noted, for example " alkaryl, wherein aryl replaces alternatively with halogen or hydroxyl ".

Term " replacement " is meant that " replacement " part comprises that 1 to 3 is selected from following substituting group: halogen, alkyl, haloalkyl, amine, nitrile, nitro, alkenyl, alkynyl, alkylthio and trinitride.

Term " treatment " is intended to comprise preventative (prophylactic) and at the therapy of (symptomatic) pattern of symptom.

Compound described herein can comprise one or more asymmetric centers, and thereby can produce diastereomer and optically active isomer.The present invention includes all this type of possible diastereomer and their racemic mixtures, they pure basically, all possible geometrical isomer, and pharmacy acceptable salt through resolved enantiomers.Above-mentioned formula is not presented at the stereochemistry of determining of some position.The present invention includes all steric isomers of described compound and pharmacy acceptable salt thereof.In addition, the mixture and the isolating particular stereoisomer that also comprise steric isomer.In the synthesis procedure process that is used for preparing this compounds, perhaps in using racemization well known by persons skilled in the art or epimerization operation, the product of this type of operation can be the mixture of steric isomer.

Term " pharmacy acceptable salt " is meant from the salt of pharmaceutically acceptable avirulent alkali or acid preparation.When described compound comprised acidic functionality, its corresponding salt can comprise mineral alkali and organic bases easily from pharmaceutically acceptable avirulent alkali preparation.The salt that is derived from this type of mineral alkali comprises aluminium salt, ammonium salt, calcium salt, copper (cupric and monovalence copper) salt, trivalent iron salt, divalent iron salt, lithium salts, magnesium salts, manganese (manganic and bivalent manganese) salt, sylvite, sodium salt, zinc salt etc.Particularly preferably be ammonium salt, calcium salt, magnesium salts, sylvite and sodium salt.

The salt that is derived from pharmaceutically acceptable avirulent organic bases comprises primary, secondary, tertiary amine, and the salt of cyclammonium and the amine that is substituted (for example, natural existence and synthetic are substituted amine).Other pharmaceutically acceptable avirulent organic basess (therefrom can form salt) comprise ion exchange resin, arginine for example, trimethyl-glycine, caffeine, choline, N, N '-dibenzyl-ethylenediamin, diethylamine, the 2-DEAE diethylaminoethanol, the 2-dimethylaminoethanol, thanomin, quadrol, N-ethylmorpholine, N-ethylpiperidine, glucamine, glycosamine, Histidine, breathe out amine (hydrabamine), Isopropylamine, Methionin, meglumine (methylglucamine), morpholine, piperazine, piperidines, versamid 900, PROCAINE HCL, PHARMA GRADE, purine, Theobromine, triethylamine, Trimethylamine 99, tripropyl amine, Trometamol, or the like.

When described compound comprises basic group for example during amine groups, its corresponding salt can comprise mineral acid and organic acid easily from pharmaceutically acceptable avirulent acid preparation.This type of acid comprises, for example acetate, Phenylsulfonic acid, phenylformic acid, camphorsulfonic acid, citric acid, ethyl sulfonic acid, fumaric acid, glyconic acid, L-glutamic acid, Hydrogen bromide, hydrochloric acid, hydroxyethylsulfonic acid, lactic acid, toxilic acid, oxysuccinic acid, amygdalic acid, methylsulfonic acid, glactaric acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succsinic acid, sulfuric acid, tartrate, tosic acid, or the like.

The I.PDE4 inhibitor

3 ', 5 '-cyclic nucleotide phosphodiesterase (PDEs) is a big fermentoid, comprises at least 11 different families, they differ from one another on structure, biological chemistry and pharmacology.Enzyme in each family is commonly called isozyme.Compound specificity ground described herein is in conjunction with in four kinds of gene products of the 4th family of PDEs one or more, i.e. PDE4A, PDE4B, PDE4C and PDE4D.

In some embodiments, described compound is nonselective and in other embodiments for these four kinds of receptor subtypes, and described compound selective ground is in conjunction with one of these hypotypes.Described therein compound is not to want to show described compound specificity ground in conjunction with the PDE4D receptor subtype in conjunction with shown data among the work embodiment of PDE4D, but shows that combining of described compound and PDE4 acceptor surpasses and the combining of other PDE acceptor types.The bonded comparative studies of described compound and other PDE4 receptor subtypes shows, combines with the PDE4D receptor subtype and have small difference aspect other PDE4 hypotypes combine at described compound.For example, the IC of compound 1 inhibition PDE4A, 4B, 4C and 4D described herein ₅₀Value be respectively 99+/-15,35+/-5,127+/-17 and 18+/-1nM.

These enzymes be known as the jointly isotype or the hypotype of PDE4 isozyme family.PDE4s is characterised in that optionally, high-affinity ground hydrolytic deterioration second messenger cyclic nucleotide, cyclic amp (cAMP); And for inhibition sensitivity by rolipram.

Usually, described compound falls into following formula:

Formula 1 formula 2 formulas 3

Wherein,

R ₁Be haloalkyl, for example methyl fluoride, difluoromethyl and trifluoromethyl,

R ₂Be C _3-6The heterocyclic radical part of the cycloalkyl of cycloalkyl, replacement, heterocyclic radical or replacement comprises cyclopentyl, tetrahydrofuran base and oxa-cyclohexyl part especially,

R ₃Be selected from C _2-10Alkyl or haloalkyl, for example C _2-5Alkyl, more particularly C ₄Alkyl comprises normal-butyl, isobutyl-and the tertiary butyl,

R ₄And R ₅Be independently selected from C _2-6Alkyl, C _3-6Cycloalkyl, haloalkyl, perhaps both be combined to form carbonyl, imines (that is ,=NH) or the thiocarbonyl part,

X, X ' and X " are independently selected from N, C-H, C-F, C-Cl, C-Br, C-I and C-R ₆,

Y is O, S or NH,

Wherein, R ₆And R ₇Hydrogen, C respectively do for oneself _2-6Alkyl, C _6-10Aryl ,-C _1-10Alkyl-C _6-10Aryl ,-C _6-10Aryl-C _1-10Alkyl, heteroaryl and

N is 1 to 10 integer.

Suitable R ₂The example of part comprises:

Wherein Y as defined above.

More particularly, R ₂Be selected from:

In one embodiment, R ₆Be

In one embodiment, described compound is defined by following formula:

Formula 4 formulas 5 formulas 6

Wherein, R _1-6, " and Y as above defines for X, X ', X.

In another embodiment, described compound is defined by following formula:

Formula 7 formulas 8 formulas 9

Wherein, R _1-6As above define with Y.

II. synthetic method

Described compound can for example prepare according to the following general method of hereinafter being summarized in flow process 1 and 2.

Synthesizing of new selective PDE4 inhibitor

Flow process 1a) R ₁(CH ₂F): CCIF ₂COOMe, Cs ₂CO ₃, DMF; B) (i) R ₂OH (3a-f), PPh ₃, t-BuO ₂CN=NCO ₂Bu, THF; (ii) R Br (3g.3h), CsCO ₃, DMF; C) R ₃Li or R ₃MgBr, THF ,-78 ℃; D) PCC, DCM

Flow process 2a) (i) R ₂OH (7a-d), PPh ₃, t-BuO ₂CN=NCO ₂T-Bu, THF; (ii) R ₂Br (7e), CsCO ₃, DMF; B) R ₃Li or R ₃MgBr, THF ,-78 ℃; C) PCC, DCM; D) R ₃Br, n-BuLi, THF ,-78 ℃; E) AcOH, H ₂SO ₄, 80 ℃; H ₂Pd/C; F) SOCl ₂, DCM; Methyl aceto acetate, CH ₃ONa, THF; LiOH, THF/MeOH/H ₂O

Because R ₁-R ₄A large amount of may the making up of group, the description in the flow process 1 provide the general synthetic of these analogues.It hereinafter is the generality discussion of this synthetic method.Hereinafter,, in these reference, described chemical reaction and condition, and described chemistry is applicable to hereinafter described compound is provided though in listed reference, do not describe definite intermediate.

From 3, the 4-Dihydroxy benzaldehyde begins, according to about with the definite document operation of chlorine difluoroacetic acid methyl esters reaction (people such as Guay, Bioorg.﹠amp; Med.Chem.Lett.12,1457-1461 (2002)) can prepare list-difluoro methyl ether (2a).List-methyl ether (2b) is obtained commercially.Can be according to similar document operation, adopt in tetrahydrofuran (THF) (THF) and R ₂The Mitsunobu of OH reacts and carries out R ₂The connection of group, thus compound 3 obtained.Have adopt Mitsunobu to react the ripe precedent for preparing phenol ether (for example, J.Chem.Soc.Perkin is (5) T.1,857-865; People such as Wang, Bioorg.﹠amp; Med.Chem.Lett.11 (7), 891-894 (2001), people such as Picard, J.Med.Chem.45 (16), 3406-3417 (2002), people such as Hirayama, Bioorg.﹠amp; Med.Chem.11 (3), 361-381 (2003), people such as Sagi, J.Med.Chem 46 (10), people such as 1845-1857 (2003) and Stelmach, Bioorg.﹠amp; Med.Chem.Lett.13 (2), 277-280 (2003); And Bioorg.﹠amp; Med.Chem.Lett.13 (12), 1997-2001 (2003)).

Work as R ₂When being aryl rings, it can be connected with the alkylation of ArBr by 2.Also have a large amount of document precedents about this type of reaction (for example, h Cherng, Y.-J.Tetrahedron 58 (24), 4931-4935 (2002), i Cherng, Y.-J.Tetrahedron 58 (24), people such as 4931-4935 (2002) and Phillips, H.Med.Chem.45,2484-2493 (2002), j LaMattina and Taylor, H.Org.Chem.46,4179-4182 (1981), with people such as Reinhard, H.Med.Chem.46 (11), 2151-2168 (2003)).In alkylation process, R ₂Amino on the OH (b, c, f) needs protected.This can finish by using Boc protect, and described Boc can be when end of synthesis handles by trifluoroacetic acid (TFA) and cuts.After two equal alkylations of phenolic hydroxyl group, can introduce R by following manner ₃Group: use alkyl lithium reagents (aryl halide prepares by handling with tert-butyl lithium), or use Grignard reagent (preparing) that the aldehyde carbonyl is carried out nucleophilic addition(Adn) by reacting with magnesium.In addition, the functional group's needs that have active proton in described aryl halide are protected.Described hydroxyl can be protected with t-butyldimethylsilyl group or tetrahydropyrans (THP) group.Described amino can be protected with the Boc group.Hydroxyl among the 4a can carry out oxidation with PCC (pyridinium chlorochromate), thereby obtains 4b, and it is a potential PDE4 inhibitor (table 2).Can replace with enolate subsequently and introduce last group (R by at first the benzyl hydroxyl of 4a being changed into the halogenide leavings group ₄), thereby obtain compound 6a (R ₄=CH ₃CO-) (

Deng the people, Bioorg.﹠amp; Med.Chem.Lett.13,741-744 (2002); With people such as Guay, Bioorg.﹠amp; Med.Chem.Lett.12,1457-1461 (2002)).Saponification and acidifying subsequently form 7a (Id.) after decarboxylation.The ketone groups of 7a can change into methylene radical (7b) by the Wolf-Kishner reduction, perhaps passes through NaBH ₄Reduction and change into alcohol (7c).Alcohol (7c) can be replaced by methyl after methylsulfonylization and the replacement of MeLi subsequently, thereby obtains 7d.Then, can carry out at R ₂And R ₃On group separately go the protection, thereby obtain final product.The alternative approach that obtains compound 7 is from 4b, carries out nucleophilic addition(Adn) by using organolithium or Grignard reagent.These two kinds of technology are all successfully used.

Should be noted that in synthetic in flow process 1 or 2 and do not have stereochemistry control.Yet, by inhibitor at first synthetic and the screening racemic form, and identify the high-affinity inhibitor, and can use chirality HPLC to come purifying and separate every kind of enantiomer to identify more effective enantiomer, begin the asymmetric synthesis of this type of enantiomer inhibitor then.For example, the compound of being discussed among the work embodiment below 1 is excellent lead compound, and it has the IC of 18nM ₅₀(table 2), but it is a racemic mixture.The alcohol of the enantiomeric pure that is obtained commercially by use can be introduced R ₂, and use chirality HPLC to separate and estimate each enantiomer.

Following raw materials according/alkylating reagent is not obtained commercially, and their preparation is described in the flow process 2.Compound 9 can prepare (people such as Brown, J.Org.Chem.50 (10), 1582-1589 (1985)) by hydroboration-oxidation of 8.14 synthetic can be from 1, the beginning of 4-two bromo-2-fluorobenzene (10) by handling with potassium tert.-butoxide and ethylene glycol, thereby obtains compound 11.11 hydroxyl can be changed into bromide.Then, on ring, carry out the lithium exchange of ortho position bromide, replace subsequently, thereby obtain 13 people such as (, Org.Lett.3,3357-3360 (2001)) Song.Further oxidation provides benzofuran compound 14 (Filler, R.Chem.Rev.63,21-43 (1963)).The synthetic of compound 16-19 can be since 15.The cyclisation in the benzene that refluxes of 3-bromobenzene oxy-aldehyde acetal and Tripyrophosphoric acid (PPA) provides compound 16 and 17 people such as (, Synth.Commun.19,257-265 (1989)) Barker.Catalytic hydrogenation should cause forming compound 18 and 19 (people such as Kuwano, Chem.Lett.4,428-429 (2000)).Synthesized intermediate 16-17.22 and 23 preparation can carry out 2 from using two suitable bromoalkanes, and the alkylation of 6-dibromophenol begins, thereby obtains aryloxy alkyl bromine 21.Then, carry out lithium exchange, carry out cyclisation subsequently, thereby provide 22 people Tetrahedron Lett.39 such as (, 2219-2222 (1998)) Kerrigan with n-Butyl Lithium.22 dehydrogenation provides compound 23 (Filler, R.Chem.Rev.63,21-43 (1963)).

III. pharmaceutical composition

Described compound can be comprised in the pharmaceutical composition.Described pharmaceutical composition comprises compound described herein or its pharmacy acceptable salt as activeconstituents and pharmaceutically acceptable carrier; And can comprise other treatment composition or auxiliary agent (adjuvant) alternatively.Optionally other therapeutic component comprises, i for example) LTRA, ii) inhibitors of leukotriene biosynthesis, iii) reflunomide, iv) H1 receptor antagonist, v) beta 2 adrenoreceptor agonists, vi) COX-2 selective depressant, vii) statin, viii) nonsteroid anti-inflammatory drugs (" NSAID "), and ix) the M2/M3 antagonist.

Described composition comprises and is suitable for the composition that oral, rectum, part and parenteral (comprising subcutaneous, intramuscular and intravenously) are used, although under given situation, only approach depends on concrete host, and character and the severity of using the symptom of described activeconstituents for it.Described pharmaceutical composition can exist with unit dosage form easily, and known any method prepares in the pharmacy field by using.

The ointment, ointment, jelly, solution or the suspension that comprise described compound can be used for local the use.For the purposes of the present invention, in the scope that use the part, comprise mouth wass and mouth wash shua.

About 0.01 dosage level to about 140mg/kg body weight/day can be used for treating following symptom: asthma for example, chronic bronchitis, chronic obstructive pulmonary disease (COPD), the eosinophilic granuloma, optimum or neoplasm dermatoses with other, endotoxin shock (and relevant symptom is for example in the laminitis and the angina of Malaysia and China), septic shock, ulcerative colitis, Crohn's disease, the reperfusion injury of cardiac muscle and brain, inflammatory arthritis, chronic glomerulonephritis, atopic dermatitis, urticaria, adult respiratory distress syndrome, chronic obstructive pulmonary disease in the animal, diabetes insipidus, allergic rhinitis, anaphylaxis conjunctivitis, vernal conjunctivitis, arterial restenosis, ortherosclerosis, atherosclerosis, neurogenic inflammation, pain, cough, rheumatoid arthritis, ankylosing spondylitis, transplant rejection and graft versus host disease (GVH disease), gastroxia, bacterium, the Sepsis of fungi or virus induction or septic shock, inflammation and cytokine mediated chronic tissue's sex change, osteoarthritis, cancer, emaciation, amyotrophy, dysthymia disorders, dysmnesia, the cancerous invasion of tumor growth and healthy tissues, they suppress to respond to PDE4; Perhaps alternatively adopt the dosage level of about 0.5mg to about 7g/ patient/sky.Can use described compound to prevent above-mentioned symptom with prevention effective dose level.

Thereby can will depend on the host that treated and specific method of application with the amount of the activeconstituents of the combined generation of solid support material single dose form and change.For example, be intended to the Orally administered preparation of giving the people and can contain about 0.5mg easily to the about promoting agent of 5g, it is mutually compound with solid support material suitable and that measure easily (can account for total composition about 5% to about 95%).Unit dosage form generally will comprise the activeconstituents of about 1mg to about 1000mg, be generally 25mg, 50mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 800mg or 1000mg.

For any specific patient, concrete dosage level will depend on series of factors, comprise age, body weight, general health, sex, diet, will use opportunity, the severity of the specified disease of route of administration, discharge rate, medication combined and experience treatment.

In fact, according to the pharmacopedics compounding technology of routine, it is combined with intimately mixed mode and pharmacopedics carrier that compound of the present invention or its pharmacy acceptable salt can be used as activeconstituents.Carrier can have various forms, and this depends on the form of the preparation that hope is used, for example oral or parenteral (comprising intravenously).Therefore, described pharmaceutical composition can be used as and is suitable for the Orally administered unit that separates and exists, for example capsule, cachet or tablet, and wherein each all contains the activeconstituents of predetermined amount.In addition, described composition can exist with following form: powder, granule, solution, the suspension in waterborne liquid, non-aqueous liquid, oil-in-water emulsion or water-in-oil liquid emulsion.Except the common formulations of showing above, described compound or its pharmacy acceptable salt also can be used by controlled release means and/or delivery apparatus.Described composition can prepare by any method in the pharmacy industry.Usually, these class methods comprise the combined step of activeconstituents and carrier (its constitute one or more must composition).Usually, described composition prepares by the solid carrier of activeconstituents and liquid vehicle or segmentation or both evenly and are nearly mixed.Then, described product can be made into desirable form easily.

Therefore, pharmaceutical composition of the present invention can comprise pharmaceutically acceptable carrier and compound or its pharmacy acceptable salt.Described compound or its pharmacy acceptable salt can also be comprised in the pharmaceutical composition with one or more other treatment active compounds combinedly.

The pharmacopedics carrier that is adopted can be for example solid, liquid or gas.The example of solid carrier comprises lactose, terra alba, sucrose, talcum, gelatin, agar, pectin, gum arabic, Magnesium Stearate and stearic acid.The example of liquid vehicle is syrup, peanut oil, sweet oil and water.The example of carrier gas comprises carbonic acid gas and nitrogen.

Be used for the composition of oral dosage form in preparation, can use any medium of pharmacopedics easily.For example, water, glycol, oil, alcohol, correctives, sanitas, tinting material etc. can be used for forming oral liquid, for example suspension, elixir and solution; And can be used for forming oral solid formulation such as the carrier of starch, sugar, Microcrystalline Cellulose, thinner, granulating agent (granulating agents), lubricant, tackiness agent, disintegrating agent etc., for example powder, capsule and tablet.Because they are used conveniently, thereby tablet and capsule be preferred oral dosage unit, adopts solid pharmacopedics carrier thus.Alternatively, tablet can carry out dressing by the water-based or the non-aqueous technology of standard.

The tablet that contains compound described herein can use one or more ancillary components or auxiliary agent alternatively by compacting or molded the preparation.Compressed tablets can prepare by the activeconstituents of compacting in suitable machine with free-flowing form (for example powder or particle), alternatively with tackiness agent, lubricant, inert diluent, tensio-active agent or dispersant.Molded tablet can prepare by the mixture of mold pressing in suitable machine with the wetting powdered compounds of inert liquid diluent.Each tablet preferably contains the activeconstituents of about 0.1mg to about 500mg; Preferably contain the activeconstituents of about 0.1mg with each cachet or capsule to about 500mg.Therefore, under the situation of taking one or two tablet, cachet or capsule (once a day, twice or three times), tablet, cachet or capsule contain the activeconstituents of 0.1mg, 1mg, 5mg, 25mg, 50mg, 100mg, 200mg, 300mg, 400mg or 500mg easily.

The pharmaceutical composition that is suitable for parenteral administration can be made into solution or the suspension of activeconstituents in water.Can comprise suitable tensio-active agent, for example hydroxypropylcellulose.Can also in glycerine, liquid polyethylene glycol and their mixture (in oil), prepare dispersion.In addition, can comprise sanitas to prevent the obnoxious growth of microorganism.

Those pharmaceutical compositions that are suitable for injecting use comprise aseptic aqueous solution or dispersion.Described composition can be the form of sterilized powder, is used for preparing immediately this type of sterile injectable solution or dispersion.In all cases, final injectable forms must be aseptic, and must be effective mobile so that can easily inject.

Described pharmaceutical composition must be stable under the condition of producing and storing; Therefore, preferably should protect at the contamination of microorganism (for example bacterium and fungi).Described carrier can be solvent or dispersion medium, comprises for example water, ethanol, polyvalent alcohol (for example, glycerine, propylene glycol and liquid polyethylene glycol), vegetables oil and suitable mixture thereof.

Described pharmaceutical composition can be for being suitable for the local form of using, for example aerosol, ointment, ointment, washing lotion, face powder etc.In addition, described composition can be the form that is suitable for using in transdermal device.Can use compound or its pharmacy acceptable salt to prepare these preparations by conventional working method.As an example, ointment and ointment prepare by following manner: mix hydrophilic material and water and the about 5wt% compound to about 10wt%, thereby produce ointment or the ointment with required denseness.

Particularly ought be used for the treatment of the irritability intestinal disorder, described pharmaceutical composition can be for being suitable for the form of rectal administration, and wherein said carrier is a solid.Preferably, described mixture forms unitary dose suppository.Suitable carriers comprises material commonly used in theobroma oil and other this areas.Suppository can form by following manner easily: at first said composition is mixed mutually cooling and shaping in mould subsequently with the carrier of remollescent or fusing.

Described pharmaceutical preparation can also comprise to washability one or more other carrier components, for example thinner, buffer reagent, correctives, tackiness agent, tensio-active agent, thickening material, lubricant, sanitas (comprising antioxidant) or the like in due course.In addition, can comprise other auxiliary agents so that described preparation and target receptor's blood etc. ooze.

IV. methods of treatment

Show that selectivity PDE4s inhibitor is effective in the various diseases model.Referring to, people such as Torphy for example, Environ.Health Perspect.102 Suppl.10:79-84,1994; People such as Duplantier, J.Med.Chem.39:120-125,1996; People such as Schneider, Pharmacol.Biochem.Behav.50:211-217,1995; Banner and Page, Br.J.Pharmacol.114:93-98,1995; People such as Barnette, J.Pharmacol.Exp.Ther.273:674-679,1995; People such as Wright, Can.J.Physiol.Pharmacol.75:1001-1008,1997; People such as Manabe, Eur.J.Pharmacol.332:97-107,1997; With people such as Ukita, J.Med.Chem.42:1088-1099,1999, the content of each piece document is integrated with this paper by mentioning in these documents.

Because described compound is the PDE4 inhibitor, so known those illnesss for the treatment of with the PDE4 inhibitor can be used compounds for treating described herein in this area.For example, described compound can be used for treating following illness by the The compounds of this invention of using significant quantity: asthma for example, chronic bronchitis, chronic obstructive pulmonary disease (COPD), the eosinophilic granuloma, optimum or neoplasm dermatoses with other, endotoxin shock (and relevant symptom, for example in the laminitis and the angina of Malaysia and China), septic shock, ulcerative colitis, Crohn's disease, the reperfusion injury of cardiac muscle and brain, inflammatory arthritis, chronic glomerulonephritis, atopic dermatitis, urticaria, adult respiratory distress syndrome, the chronic obstructive pulmonary disease in the animal, diabetes insipidus, allergic rhinitis, anaphylaxis conjunctivitis, vernal conjunctivitis, arterial restenosis, ortherosclerosis, atherosclerosis, neurogenic inflammation, pain, cough, rheumatoid arthritis, ankylosing spondylitis, transplant rejection and graft versus host disease (GVH disease), gastroxia, bacterium, the Sepsis of fungi or virus induction or septic shock, inflammation and cytokine mediated chronic tissue's sex change, osteoarthritis, cancer, emaciation, amyotrophy, dysthymia disorders, dysmnesia, the cancerous invasion of tumor growth and healthy tissues, thereby they be can be by the cAMP level of the rising that suppresses the PDE4 isozyme and cause improved disease.

In addition, as mentioned above, compound of the present invention can be united use with the other treatment compound.Especially, PDE4 inhibition combination of compounds of the present invention can advantageously be united use with following material: i) LTRA, ii) inhibitors of leukotriene biosynthesis, or iii) M2/M3 antagonist.

Selectivity PDE4 inhibitor can be used for the treatment of many inflammatories, respiratory system and anaphylactic disease and symptom, for example asthma; Chronic obstructive pulmonary disease (COPD) comprises chronic bronchitis, pulmonary emphysema and bronchiectasis; Chronic rhinitis; And chronic sinusitis.In the patient's who suffers from asthma and other obstructive airway diseases air flue, as the target that is used for drug discovery, PDE4 is most important in the PDE isozyme, because it is distributed in airway smooth muscle and the inflammatory cell.

Airflow obstruction and airway inflammation are the features of asthma and COPD.Selectivity PDE4 inhibitor can be used for treating these illnesss, flows in the lung of the animal that is subjected to the anaphylactogen attack because they have reduced eosinophilic granulocyte, and has reduced at anaphylactogen and attacked the bronchoconstriction of back appearance and the segmental bronchus responsiveness of increase.The PDE4 inhibitor also suppresses activity of immune cells, comprises CD4 ⁺T-lymphocyte, monocyte, mastocyte and basophilic granulocyte; Reduce pulmonary edema; Suppress excitability non-adrenergic non-cholinergic neurotransmission (eNANC); Strengthen inhibitory non-adrenergic-cholinergic neurotransmission (iNANC); Reducing airway smooth muscle mitotic division takes place; With induce bronchiectasis.The PDE4 inhibitor also suppresses the activity of many inflammatory cells relevant with the physiopathology of COPD, comprises monocyte/macrophage, CD8 ⁺T-lymphocyte and neutrophilic granulocyte.The PDE4 inhibitor also reduces vascular smooth muscle mitotic division to be taken place, and disturbs the airway epithelia cell to produce the ability of short scorching amboceptor effectively.By discharging neutral protease and acid hydrolase from their particle, and produce the active oxygen classification, neutrophilic granulocyte helps the disorganization relevant with chronic inflammatory diseases, and further involves the pathology such as emophysematous symptom.

The PDE4 inhibitor can be used for by improving cAMP, and suppress super-oxide generation, threshing, chemotaxis and tumor necrosis factor alpha (TNF α) release (in eosinophilic granulocyte, neutrophilic granulocyte and monocyte) and treat obstructive pulmonary disease, comprise COPD.

Vomiting is promptly felt sick and gastric disorder causing nausea, be the disadvantageous side effect relevant, and be observed under these circumstances with some PDE4 inhibitor, promptly wherein at first with regard to the CNS indication for example dysthymia disorders the PDE4 inhibitor is studied.Therefore, in one embodiment, just can not pass hemato encephalic barrier effectively and select these compounds.Producing too much hydrochloric acid in gastric juice can be another disadvantageous side effect of PDE4 inhibitor.

By selecting impermeable compound of going into central nervous system, perhaps, can minimize or avoid above-mentioned relevant with the PDE4 inhibitor sometimes adverse events by through sucking rather than orally using described compound.

About the PDE4 hypotype, A, B, C and D have found that PDE4C is more insensitive for all inhibitor usually; And, also do not have the inhibitor specificity (to be defined in IC so far about hypotype A, B and D ₅₀10 times of differences of value aspect) definite evidence.Hint in this area, at S _rThe PDE4 inhibitor that the site has high-affinity may be associated with vomiting and the gastric acid secretion that increases.

Can in animal model, estimate the PDE4 inhibitor, the PDE4 of described animal model proof PDE4 inhibitor suppresses active, for example, the bronchoconstriction that suppresses antigen induction, as people such as Cavalla, Amer.J.Respir.Crit.Care Med., disclosed among the 155:A660 (1997), the content of the document is integrated with this paper by mentioning.

Also studied the PDE4 inhibitor is used for the treatment of Crohn's disease, a kind of chronic granuloma inflammatory diseases that is usually directed to terminal ileum of the cause of disease the unknown cicatrization of intestines wall wherein occurs and thickens, and this usually causes the formation of intestinal obstruction and fistula and abscess.

Also made much to make great efforts to improve the selectivity (above further describe) of PDE4 inhibitor in the art about A to D hypotype.There is the PDE4 isozyme of four kinds of known isotypes (hypotype) at present, comprises seven kinds of splice variants (also further describing as mentioned).PDE4D (PDE4B) isotype mRNA for example expresses in neutrophilic granulocyte and the eosinophilic granulocyte at inflammatory cell, and hints in this area, and the clinical efficacy that the B-selective depressant of PDE4 can provide has the side effect of minimizing simultaneously.

Find in the publication that can get in the art about the further background knowledge of selectivity PDE4 isozyme, Norman for example, " PDE4 inhibitors 1999, " Exp.Opin.Ther.Patents 9 (8): 1101-1118,1999 (Ashley Publications Ltd.); And Dyke and Montana, Exp.Opin.Invest.Drugs 8 (9) 1301-1325,1999 (Ashley Publications Ltd.).

Treatment is used and clinical endpoint (Clinical Endpoints)

The treatment that provides compound described herein can be used for is below used, and use the explanation of relevant clinical endpoint and various external test method and animal model experiment (it can provide the data of the treatment effectiveness that is enough to define and prove described compound) with this type of treatment.

The treatment effectiveness of described compound can be applicable to patient or experimenter, and described patient or experimenter suffer from disease shown in this article or symptom and therefore need such treatment.Useful result is that to be administered to animal or to be administered to the people all be curative.

Described compound suppresses the PDE4 isozyme and therefore has the treatment application (as hereinafter further describing) of wide region, because the isozyme of PDE4 family plays an important role in all mammiferous physiology.The enzymatic action of being carried out by the PDE4 isozyme is hydrolysis in the cell of cyclic amp (cAMP) among short scorching white corpuscle.And cAMP is responsible for mediating the effect in vivo of many hormones, and thereby PDE4 be suppressed in the various physiological processes and play an important role.Have a large amount of documents in the prior art, they have described the effect that the PDE inhibitor is replied for various inflammatory cells, and it comprises also that except cAMP raises suppressing super-oxide generation, threshing, chemotaxis and tumour necrosis factor (TNF) discharges (in eosinophilic granulocyte, neutrophilic granulocyte and monocyte).

The isozyme of PDE4 family is the principal mode of the phosphodiesterase found in involving the cell type of chronic inflammatory disease, and in the bone marrow derived cell type, has only thrombocyte not express PDE.PDE4 is the enzyme of main metabolism cAMP in immunity and inflammatory cell, and is one of the enzyme of two kinds of main metabolism cAMP in airway smooth muscle.PDE4 exclusively is present in neutrophilic granulocyte, eosinophilic granulocyte, basophilic granulocyte and the monocyte.Used experiment in vitro to prove the useful anti-inflammatory action of PDE inhibitor in the past, described experiment in vitro determines that the superoxide that this compounds suppresses in person monocytic cell, eosinophilic granulocyte and the neutrophilic granulocyte produces; Amboceptor in basophilic granulocyte, scavenger cell and the neutrophilic granulocyte discharges; Discharge with the TNF α in monocyte and the scavenger cell.The amboceptor that the PDE inhibitor also suppresses inflammatory cell such as monocyte and monocyte derived scavenger cell, pulmonary mastocyte, T lymphocyte, bone-marrow-derived lymphocyte, pulmonary alveolar macrophage and eosinophilic granulocyte discharges.

Described compound provides useful anti-inflammatory action, because the PDE4 inhibitor effectively suppresses to discharge from the TNF α of mononuclear phagocyte.

Asthma

PDE4 inhibitor, particularly PDE4B inhibitor can be used for the treatment of asthma, comprise atopy and ergotropy asthma.As used herein, term " atopic asthma " is intended to and " allergic asthma " synonym, i.e. the bronchial asthma that shows as the allergy in the people of sensitization; As used herein, " ergotropy asthma " is intended to refer to every other asthma, the especially special property sent out or " very " asthma, and it is caused by various factors, comprises strenuous exercise, pungency particle, stress etc.

These compounds can use following art-recognized model to prove in the validity aspect treatment atopy or the ergotropy asthma: PDE suppresses, and the eosinophilic granulocyte activatory suppresses and the cellular infiltration model.Representative assay method based on the use primates is described in people such as Turner, Inflammation Research 45:239-245, and 1996, the content of the document is integrated with this paper by mentioning.The anti-inflammatory activity of described compound can also suppress to prove by the eosinophilic granulocyte activatory, as what measure by the LTE4 generation that is stimulated by the SEPHADEX globule in people's whole blood.

In view of their anti-inflammatory activity, they suppress relevant characteristic with them with the PDE isozyme for the influence of airway hyperreactivity, and particularly as selectivity PDE4 inhibitor, described compound can be used for treatment and prevention obstructive and airway inflammatory disease.Therefore, protect in advance with at the bronchoconstriction that causes because of obstructive or airway inflammatory disease or the recurrence of other symptomatic outbreaks by in the period that prolongs, using described compound continuously and regularly, can providing.Described compound can be used as bronchodilator to be used for the treatment of chronic or acute bronchoconstriction and being used at symptom ground treatment obstructive and airway inflammatory disease.

Obstructive and airway inflammatory disease that the present invention can be applicable to comprise: asthma; Pneumoconiosis; Chronic eosinophilic pneumonia; Chronic obstructive air flue or lung disease (COAD or COPD); With adult respiratory distress syndrome (ARDS), and by the aggravation of the caused airway hyperreactivity of other drug therapy (for example, acetylsalicylic acid or beta-2-agonists therapy).

Described compound also can be used for treating any kind, the cause of disease or pathogenetic pneumoconiosis, comprises for example aluminosis (bauxit workers disease); Anthracosis (miners' asthma); Asbestosis (steam fitter's asthma); Chalicosis (silicosis); The ostrich hair pneumoconiosis that causes owing to the dust that sucks from the ostrich feather; Owing to sucking the arc-welder's disease that the iron particle causes; Silicosis (grinder's disease); Byssinosis (flocking asthma); And talc pneumoconiosis.

Chronic obstructive pulmonary disease (COPD)

Described compound can be used for the treatment of COPD or chronic obstructive airway disease (COAD), comprises chronic bronchitis, pulmonary emphysema or associated expiratory dyspnea.COPD is characterised in that irreversible progressive obstruction of the air passage.The hyperplasia of submucosal mucus secretion sexual gland body and loose relevant in chronic bronchitis and the big cartilage air flue.Emophysematously be characterised in that the destruction of alveolus wall and the forfeiture of lung elastance.

The same with asthma, COPD is feature with the inflammation, but the inflammatory cell of finding in patient's bronchoalveolar lavage fluid and phlegm is neutrophilic granulocyte rather than eosinophilic granulocyte.In COPD patient, also find the inflammatory mediators level of rising, comprise IL-8, LTB ₄With TNF-α, and this type of patient's bronchial superficial epithelium and propria lamina (sub-epithelium) are found by T-lymphocyte and macrophages infiltration.

Bronchitis and bronchiectasis

Described compound can be used for the treatment of any kind, the cause of disease or pathogenetic bronchitis, comprises acute bronchitis, acute laryngotracheobronchitis, arachidic bronchitis, catarrhal bronchitis, chronic bronchitis, croupous bronchitis, dry bronchitis, infective asthma bronchitis, productive bronchitis and alveolar shape bronchitis.

Described compound is treated the validity of bronchial asthma, chronic bronchitis and relative disease and illness as bronchodilator or bronchial spasmolytic agent, can use that animal model proves in many different bodies known in the art.

Can for example produce the anti-inflammatory activity that proves described compound by the TNF α that has suppressed in people's whole blood, to stimulate by lipopolysaccharides (LPS).

The rhinitis of supersensitivity and other types; Sinusitis paranasal sinusitis

Allergic rhinitis is a feature with nasal obstruction, itch, watery rhinorrhea, sneeze and anosmia once in a while.Allergic rhinitis is divided into two kinds of disease categories, and is promptly seasonal and secular, and wherein the former is owing to pollen or outdoor mould spores, and the latter is owing to common anaphylactogen, for example house dust mite, animal scurf and mould spores.Allergic rhinitis generally shows replys and late phase responses in early days.Reply relevantly with the mastocyte threshing in early days, and late phase responses is a feature with the lymphocytic infiltration of eosinophilic granulocyte, basophilic granulocyte, monocyte and T-.These cells also discharge various inflammatory mediators, and all these amboceptors can be facilitated the inflammation that is shown in replying late.

According to the cause of disease and pathogenesis approaching and that share in some cases on the anatomy, sinusitis paranasal sinusitis is relevant with rhinitis.Sinusitis paranasal sinusitis is the inflammation of hole, and this symptom can be suppurative or apyogenous, and can be acute or chronic.

Because the PDE4 inhibitor is effectively at the treatment aspect of inflammation, so compound described herein can be used for the treatment of rhinitis and sinusitis paranasal sinusitis.

Rheumatoid arthritis, osteoarthritis, pain, heating and gout

Inflammation and rheumatoid arthritis that sacroiliitis is defined as the joint are the chronic systemic diseases that is mainly the joint, are characterized in inflammatory variation and the myatrophy and the osteoporosis of synovial membrane and articulation structure.The characteristics of the late stage of rheumatoid arthritis are ankylosis and distortion.As used herein, term " rheumatoid arthritis " is intended to comprise the sacroiliitis form of being correlated with and interrelating well-known in the art, for example acute arthritis, chronic inflammatory arthritis, degenerative arthritis, infective arthritis, Lyme arthritis, proliferative arthritis and vertebral joint inflammation.

Three main pathological characteristicses being responsible for the rheumatoid arthritis of carrying out property destruction of joint are inflammation, unusual cell and humoral response and synovial hyperplasia.Pro-inflammatory cytokine, for example IL-1, IL-4, IL-5, IL-6, IL-9, IL-13 and TNF-α are the main contribution factors that causes joint tissue damage, inflammation, hyperplasia, pannus formation and bone resorption.Referring to Firestein and Zvaifier, Arth.Rheum.33:768-773,1990.

PDE4 inhibitor described herein can be used for treating rheumatoid arthritis, because they can suppress the various inflammatory cells activity of (comprising basophilic granulocyte, eosinophilic granulocyte and mastocyte).Described compound can be used for treating rheumatoid arthritis, also because they are in the validity that suppresses aspect the T-cell proliferation of many different factors (comprising antigen) mediation.Described compound can promote monocyte to discharge cytokine IL-10, and it can reduce synovial fluid mononuclear cells generation TNF-α, IL-1, IL-4, IL-5, IL-6, IL-9, IL-13 and GM-CSF, and this has further strengthened total anti-inflammatory property of PDE4 inhibitor.In addition, described compound can be suppressed to be associated with inflammatory animal model from the ability of the release of the monocyte through stimulating TNF-α, anti-inflammatory action can be shown as the inhibition corresponding to TNF-α accumulation in described model.

Rheumatoid arthritis animal model as known in the art has proved, the PDE4 inhibitor can be used to assess the validity of compound described herein for regulating in the body of TNF-α and they are used for the treatment of dependency between the effectiveness of rheumatoid arthritis.A kind of this type of animal model is mouse adjuvant arthritis (adjuvant arthritis) model, as people such as Sekut, described in Olin.Exp.Immunol.100 (1): the 126-132 (1995).

Gout

Gout is meant one group of disorder of purine metabolism, and Fa Zhan gout shows as the various combinations of following phenomenon fully: hyperuricemia, the recurrent, the characteristic acute inflammation sacroiliitis that cause by the crystal of monosodium urate monohydrate, described crystal is in extremities joint or uratoma deposition on every side (tophaceous deposits) (this can cause destruction of joint and seriously disable), and uric acid urolithiasis.

Because described compound inflammation-inhibiting is so they can also be used for the treatment of gout and heating and the pain relevant with inflammation.

The illness relevant with eosinophilic granulocyte

As the part of its overall anti-inflammatory activity, the PDE4 inhibitor can suppress the eosinophilic granulocyte activation.Therefore, described compound can be used for the treatment of conditions treatment relevant with eosinophilic granulocyte.This type of illness comprises the eosinophilia, and it is to form in blood and the unusual a large amount of eosinophilic granulocyte of accumulation.Can use compounds for treating eosinophilia lung described herein to soak into, chronic eosinophilic pneumonia, and the symptom of tropical lung eosinophilia and bronchopneumonic aspergillosis (using antimicrobial therapy to treat the basic reason of these illnesss best).

Term " granulomatous (granulomatous) " expression " contains granuloma ", and term " granuloma " is meant that the monokaryon inflammatory cell of any little tubercular demarcation assembles, or be similar to this type of set of epithelial modified scavenger cell, usually round a circle lymphocyte, have common fibrosis around infringement around it.Some granuloma comprises eosinophilic granulocyte.Granuloma forms and has represented by various infectivities and the initial chronic inflammatory reaction of the non-infectious factor.Many these type of granulomatous symptom, for example allergic granulomatous vasculitis (being also referred to as Qiu-Si syndrome), polyarteritis nodosa (PAN) can use compounds for treating described herein with other illnesss relevant with eosinophilic granulocyte.

Atopic dermatitis, urticaria, conjunctivitis and uveitis

Atopic dermatitis is a kind of chronic inflammatory skin disorder, and it sees has in the individuality that tends to the hereditary predisposition that reduces for the skin thresholding of pruritus, often with allergic rhinitis, spring fever and asthma, and is feature with the extreme itch mainly.Atopic dermatitis is also referred to as allergic dermatitis and supersensitivity or atopic eczema.Atopic dermatitis usually links together with asthma and anaphylaxis, therefore can treat with the PDE4 inhibitor.

The PDE4 inhibitor can be used for the treatment of the tetter that is driven by eosinophilic granulocyte, usually by topical application.The PDE4 inhibitor can be used for the treatment of atopic dermatitis, vasodilation and urticaria (comprising cholinergic urticaria, cold urticaria, contact urticaria and strophulus), and various ocular disorders, for example conjunctivitis and uveitis.

Multiple sclerosis and other inflammatory autoimmune diseases

Multiple sclerosis is a kind of autoimmune disorder, and except chronic inflammatory diseases and demyelination, this illness also causes the gliosis in central nervous system.In multiple sclerosis, inflammatory damage is confined to but spread all over the white matter of central nervous system in the white matter of central nervous system, though be characterised in that the sign of the harden plaque of demyelination for this disease.And the development of demyelination is caused by the oligodendrocyte necrosis, and demyelination is relevant with the instillation of mainly being made up of T-cell and scavenger cell, and described T-cell and scavenger cell are expressed II class major histocompatibility complex (MHC) with local cell (for example astroglia cell, microglia and capillary blood vessel brain endothelial cell).Therefore, these cells involve antigen presentation and inflammatory reaction, and in the cerebral tissue of multiple sclerosis patients, identified many pro-inflammatory cytokines, comprised that TNF-α, TNF-β, IL-1, IL-6 and IFN-γ are being found, and their existence is generally relevant with active infringement.

The compound that suppresses TNF-α generation has become focus, because TNF-α induces astroglia cell to express the surface adhesion molecule, with relevant with disintegrating of hemato encephalic barrier in external mediation myelin and oligodendrocyte damage.Animal model has been used for proving the effect of TNF-α, for example in experimental allergic encephalomyelitis (EAE), has used anti-TNF antibody or soluble TNF acceptor and demonstrate the protectiveness effect is provided at multiple sclerosis.Reported the directly related property between the progress of TNF-α mRNA level and EAE.Referring to people such as Reeno, J.Immunol.154:944-953,1995.

Because the PDE4 inhibitor reduces TNF-α, so they can be used for the treatment of multiple sclerosis.Except suppressing PDE4 activity and TNF-α generation, described compound also serves as immunosuppressor, and can be used for the treatment of the autoimmune disease relevant with inflammation.

Other the autoimmune disease/inflammatory diseasess that can be treated include, but not limited to autoimmunity hematology illness, for example hemolytic anemia, aplastic anemia, pure red cell anaemia and idiopathic thrombocytopenic purpura; Systemic lupus erythematosus; Polychondritis; Scleroderma; Wegner granulomatosis; Dermatomyositis; Chronic active hepatitis; Myasthenia gravis; Si-Yue syndrome; The special property sent out sprue; Autoimmunity inflammatory bowel, for example ulcerative colitis and Crohn's disease; Endocrine ophthalmopathy; Graves disease; Sarcoidosis; Pulmonary alveolitis; Chronic hypersensitivity pneumonia; Primary biliary cirrhosis; Juvenile onset diabetes (type i diabetes); Anterior uveitis and granulomatous (back) uveitis; Keratoconjunctivitis sicca and epidemic keratoconjunctivitis; Diffuse interstitial pulmonary fibrosis (a matter pulmonary fibrosis); Idiopathic pulmonary fibrosis; Cystic fibrosis; Psoriatic arthritis; There is and do not have the glomerulonephritis of nephrotic syndrome, comprise acute glomerulonephritis, idiopathic nephrotic syndrome and MCN; Inflammatory/hyperplasia dermatoses comprises psoriatic and atopic dermatitis (further going through as mentioned), contact dermatitis, allergic contact dermatitis, benign familial pemphigus, pemphigus erythematosus, pemphigus foliaceus and pemphigus vulgaris.

In addition, described compound also can be used as immunosuppressor to be used for the allograft rejection reaction behind the prevention of organ transplant, and wherein this organoid generally includes the tissue from marrow, intestines, heart, kidney, liver, lung, pancreas, skin and cornea.

Inflammatory bowel

Ulcerative colitis (UC) is a kind of chronic recurrent ulcer in colon, mainly occur in mucous membrane and submucosa, it is agnogenio, and its clinical manifestation is spastic abdominal pain, proctorrhagia, and the blood, purulence and the mucous loose ejecta that have few ight soil particulate.The relative disease of intestines comprises collagenous colitis, colitis polyposa and transmural colitis.Crohn's disease (CD) is agnogenic chronic granuloma inflammatory diseases, it relates to GI any part, but be usually directed to have the cicatrization of intestines wall and the terminal ileum that thickens, this usually causes the formation of intestinal obstruction and fistula and abscess, and has the high relapse rate after treatment.Ulcerative colitis, Crohn's disease and the relative disease of above being discussed are called as inflammatory bowel (IBD) together.

The therapy that is used for inflammatory bowel at present comprises 5-aminosalicylic acid, reflunomide and immunomodulator, for example azathioprine, Ismipur and methotrexate, and they are used is because their suppress the ability that TNF-α produces.Selectivity PDE4 inhibitor, compound for example described herein suppresses to discharge TNF-α from peripheral blood lymphocytes, therefore can be used for the treatment of inflammatory bowel.

Septic shock, renal failure, emaciation and infection

Septic shock is and utmost point severe infection, and the relevant shock of gram-negative bacterial infections the most normally is although it can be produced by other bacteriums, virus, fungi and protozoon.Septic shock is considered to result from the effect of other products of intracellular toxin or infectant for vascular system, and it causes a large amount of blood to be isolated in capillary vessel and the vein.Also relate to the activation of complement and kinin system, and the release of histamine, cytokine, prostaglandin(PG) and other amboceptors.

Renal failure is that kidney can not be drained the metabolite of normal plasma level under normal load conditions, perhaps can not keep ionogen under normal absorption condition.In the rat model of the acute renal failure of endotaxin induction, selectivity PDE4 inhibitor demonstrates remarkable increase urine cAMP and drains, and obviously weakens the increase of the renal vascular resistance of endotaxin induction, and reduces renal blood flow and glomerular filtration rate(GFR.Referring to people such as Carcillo, Pharmacol.Exp.Ther.279:1197 (1996).Therefore, compound described herein as selectivity PDE4 inhibitor, can be used for the treatment of renal failure, for example acute renal failure.Emaciation be with overall unhealthy and malnutritive be the constitutional disease of feature.Emaciation can be caused by following factors: the forfeiture of malaria, body fluid or other organ dysfunctions or decline, late period renal failure, heart trouble, ypotension, weight loss, anorexia, adrenocortical hormone shortage, tuberculosis, cancer, human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) (AIDS).Described compound can be used for treating emaciation, discharges because they can reduce or suppress TNF-α.

Described compound can also be used for the treatment of the symptom of infection, and the product that wherein said infection increases TNF-α among the patient becomes.The example of such infection comprises virus infection, for example HIV-1, HIV-2 and HIV-3; Cytomegalovirus (CMV); Influenza; Adenovirus; And simplexvirus, particularly zoster and herpes simplex.

Described compound can also be used for the treatment of the symptom of yeast and fungi infestation, and wherein said yeast and fungi are responsive or cause that the TNF-α among the patient produces for the rise by TNF-α.

When being used for the treatment of the symptom of infection, described compound can and should be used with biocide, for example, and polymyxins, for example PXB; Imidazoles, for example clotrimazole, econazole, miconazole and KETOKONAZOL; Triazole species, for example fluconazole and itraconazole; With the amphotericin class, for example amphotericin B and AM Bison.

Pulmonary hypertension

Phosphodiester enzymic hydrolysis vasorelaxation second messenger cAMP and cGMP, and increase by the pulmonary hypertension (HPH) by hypoxia inducible.Anoxic is to organize oxygen supply to be reduced to be lower than physiological level, although blood is enough to the perfusion of tissue.The pulmonary hypertension that the result causes is characterised in that the pressure within the pulmonary artery circulation raises, and, is higher than the systolic pressure and the diastolic pressure that is higher than 12mm Hg of 30mm Hg that is.

Using from normal rat and in from the rat model that has by the isolating pulmonic ring of the rat of the pulmonary hypertension of hypoxia inducible, selectivity PDE4 inhibitor demonstrates the activity of relaxing that has strengthened Racemic isoproterenol and Forskolin.Therefore, the PDE4 inhibitor can significantly improve pulmonary artery and relaxes in the pulmonary hypertension by hypoxia inducible.Therefore, described compound can be used for treating pulmonary hypertension, particularly by the pulmonary hypertension of hypoxia inducible.

The CNS illness

Known selectivity PDE4 inhibitor provides useful effect in dysthymia disorders and other central nervous system disorders, comprise Parkinson's disease people such as (, Eur.H.Neurosci.7:2431-2440,1995) Hulley; And learning and memory obstacle (people such as Egawa, Jpn.J.Pharmacol.75:275-281,1997).The PDE4 inhibitor also has been used for the treatment of tardive dyskinesia and pharmacological dependence (WO 95/28177 and JP 92221423 (1997)).Found that also the PDE4 isozyme plays main effect aspect the Dopamine HCL biosynthesizing in the control midbrain neuron.Therefore, the PDE4 inhibitor, compound for example described herein, can be used for the treatment of with midbrain neuron within and on every side Dopamine HCL is relevant or by the illness and the disease of its mediation.

Described compound can also be used for the treatment of under arteriosclerotica dementia and the cortex dull-witted.Arteriosclerotica dementia also claims vascular dementia and Dementia with Multiple Brain Infarction, is so a kind of dementia, and it has the irregular distribution of the neurologic defect that causes with the process that progressively worsens of a series of transient apoplexy forms with by cerebrovascular disease.Dull-wittedly under the cortex cause, and it is characterized in that the loss of memory, be accompanied by at process information or make aspect the intelligence response slow by the infringement that influences cortex hypencephalon structure.Comprise the dementia that is accompanied by Huntington Chorea, hepatolenticular degeneration, Parkinsonism and thalamus atrophy.

Use compound described herein and composition can also treat other illness, include but not limited to unipolar depression, acute and chronic neurodegenerative disorders, Parkinson's disease, mild cognitive impairment, the loss of memory, alzheimer's disease, spinal cord injuries receptor, craniocerebral injury or multiple sclerosis with inflammatory component.

Other treatment is used

Known PDE4 inhibitor can be used for treating ischemia reperfusion injury (people such as Block, NeuroReport 8:3829-3832,1997); Autoimmune diabetes (people such as Liang, Diabetes 47:570-575,1998); Retina autoimmunity (people such as Xu, Invest.Ophthalmol.Visual Sci.40:942-950,1999); Lymphocytic leukemia (Kim and Lerner, Blood 92:2484-2494,1998); HIV infects people such as (, AIDS 9:1137-1144,1995) Angel; Lupus erythematosus (JP10067682 (1998)); Kidney and ureteral (DE 4230755 (1994)); The genito-urinary system disorder of gastrointestinal tract (transferring the WO 94/06423 of Schering AG) of unifying; And prostatosis (WO 99/02161).

Therefore, compound disclosed herein can be used for the treatment of any above-mentioned illness.

V. prove the assay method of biologic activity

Various assay methods can be used to prove the effect (for example, binding affinity, selectivity, and/or activity) of described compound.Representational example is provided below.

In people's whole blood by LPS and FMLP inductive TNF α and LTB ₄Assay method

Whole blood provides the environment of rich in proteins and cell, and it is suitable for studying the biological chemistry effect of anti-inflammatory compound (for example PDE4 selective depressant).

But the normal human blood that does not stimulate does not comprise the TNF-α and the LTB of detection level ₄After stimulating with LPS, activated monocytes justacrine TNF-α was until 8 hours, and blood plasma level keeps stablizing 24 hours.Show through disclosed research, occurring on the transcriptional level by suppressing through PDE4 and/or the enhanced adenylate cyclase activity increases the inhibition of the TNF-α that cAMP causes in the cell.

Can analyze the PDE4 inhibitor with regard to the activity that they suppress aspect LPS inductive TNF α generation in human peripheral blood mononuclear cell (PBMCs).LPS (lipopolysaccharides) can buy from Sigma.PBMCs can buy (THP-1, ATCC code T IB-202) from ATCC, and can use standard scheme to cultivate and separate by 1-STEP POLYMORPHS substratum (Accurate Chemical, Westbury NY).

Can be 2 * 10 with concentration with purified human PBMC s ⁶The concentration of individual cell/ml is suspended in RPMI 1640 substratum of the FBS (foetal calf serum) that is supplemented with 10% heat inactivation and 1% microbiotic (penicillin-Streptomycin sulphate).Aliquots containig (2 * 10 with 100 μ l ⁵Individual cell) is added in the 96 hole microtiter plates.Before lipopolysaccharides (10ng/ml) stimulates, can add PDE4 inhibitor and contrast (final concentration of DMSO is 0.1% in the substratum) with the dosage range of 0.1nM-10 μ M to described cell, and keep 60 minutes.In 37 ℃ at 5%CO ₂In the incubator after incubation 18-20 hour, can be by carrying out centrifugally collecting acellular supernatant liquor in 800g, and be stored in-80 ℃.Then, can come described supernatant liquor is analyzed, for example use by R﹠amp with regard to the amount of TNF α; D Systems (Minneapolis, MN) Kai Fa QUANTIKINE immunoassay test kit.IC ₅₀Value can use GRAPHPAD Prism software to calculate.

Antiallergic activity in the body

Described compound is tested in the influence of the supersensitivity pneumonia that can just mediate for IgE (it is to suck antigen and inductive by the cavy by sensitization).At first, can be by peritoneal injection and aluminium hydroxide and the combined antigen of pertussis vaccine, make cavy under slight endoxan inductive immunosuppression for ovalbumin sensitization.Can give antigenic booster dose after 2 and 4 week.When 6 week, can attack animal with the ovalbumin of aerosolization, and intraperitoneal is used for example Pyrilamine of antihistaminic agent simultaneously.After further 48 hours, can carry out bronchoalveolar lavage (BAL), and can count eosinophilic granulocyte and other number of WBC in BAL liquid.Can take out lung to be used for the histological examination of inflammatory damage.After using described compound, can assess, with respect to contrast, whether the accumulation of eosinophilic granulocyte and other inflammatory leukocytes significantly reduces, and/or whether less inflammatory damage is arranged in the lung of the animal through treating.

PDE activation measurement scheme based on SPA

Can followingly screen and suppress the compound that IV type cAMP specific phosphodiesterase enzyme is hydrolyzed into cAMP AMP with 96 hole flat type:

In 96 hole flat boards, add test-compound (being dissolved among the 2 μ L DMSO) in 30 ℃, 188 μ L substrate buffer solutions, its comprise [2,8- ³H] cyclic amp (cAMP, 100nM to 50 μ M), 10mM MgCl ₂, 1mM EDTA, 50mM Tris, pH 7.5.Come initial action by adding the 10 μ L people PDE4 (control described amount, thereby make in 10 minutes to form about 10% product) that recombinates.After 10 minutes, (Piscataway N.J.) comes termination reaction for AmershamPharmacia Biotech, Inc. by adding 1mg PDE-SPA pearl.At Wallac

96 hole plate count device (EG﹠amp; G Wallac Co., Gaithersburg Md.) goes up the quantitative product A MP that produces.Signal when not having enzyme is defined as background.100% activity is defined in detected signal when having enzyme and DMSO, and subtracting background.Thereby, calculate the inhibition percentage.IC ₅₀Value can be estimated from the titration of 10-point with nonlinear regression and fitting (using 4-parameter/many binding sites equation of standard).

The following example is intended to illustrate certain embodiments of the present invention, but and does not mean that and limited the present invention.Unless specify in addition, experimental arrangement carries out under following condition.All operations promptly carries out under 18-25 ℃ temperature under room temperature or envrionment temperature.Decompression (600-4000 pascal: 4.5-30mm Hg),, use Rotary Evaporators to carry out the evaporation of solvent with until 60 ℃ bath temperature.(TLC) comes the following response process by tlc, and provides only explanation for example of reaction times.Fusing point is uncorrected, and ' d ' expression is decomposed.Given fusing point is the fusing point that obtains for the material that is prepared as described.Polymorphism can cause isolating the sample with different melting points in some preparations.The structure of all the finished product and purity are guaranteed by at least a following technology: TLC, mass spectroscopy, nucleus magnetic resonance (NMR) spectral method or trace analysis data.Give only explanation for example of the rate of output.When providing, the NMR data are with the form of delta (δ) value of main diagnostic proton (major diagnostic protons), be expressed as with respect to the ppm (ppm) as interior target tetramethylsilane (TMS), it is measured under 300MHz, 400MHz or 500MHz by the solvent shown in using.The routine that is used for signal shape is abbreviated as: s., and unimodal; D., doublet; T., triplet; M., multiplet; Br., wide; Or the like.In addition, " Ar " expression aromatic signal.Chemical symbol has their common implication; Also used following abbreviation: v (volume), w (weight), b.p. (boiling point), m.p. (fusing point), L (liter), ml (milliliter), g (gram), mg (milligram), mol (mole), mmol (mmole), eq (equivalent).

To understand the present invention better with reference to following non-restrictive example.

Synthetic and the assessment of embodiment 1:PDE4 inhibitor

Use in above-mentioned flow process I and II generalized chemistry synthesize the PDE4 inhibitor of a series of supposition, and assess their inhibition activity for various PDEs.These PDEs comprise PDE4A10, PDE4B2B, PE4C2, PDE4D2, PDE2A3, PDE3A, PDE5A1, PDE7A1, PDE9A2 and PDE10A2.

The catalyst structure domain subclone of PDE2A, PDE3A, PDE4A-D, PDE5A, PDE7A, PDE9A and PDE10A is gone into also to cross expression in the intestinal bacteria therein.Subclone, cross to express and protein purification is a standard technique, and the scheme that is used for purifying PDE4D, PDE5A, PDE7A and PDE9A is disclosed (Huai, Q., Wang H, Sun, Y., Kim, H.Y., Liu, Y. and Ke, H., Structure 11,865-873 (2003); Huai, Q., Colicelli, J. and Ke, H.Biochemistry 42,13220-13226 (2003); Huai, Q., Liu, Y., Francis, S.H., Corbin, J.D. and Ke, H., J.Biol.Chem., 279,13095-13101 (2004); And Wang, H., Liu, Y., Chen, Y., Robinson, H. and Ke, H.J.Biol.Chem.280,30949-30955 (2005); People such as Huai, Proc Natl Acad Sci USA.101,9624-9629 (2004)).Therefore, hereinafter only simplified summary committed step and result.

EST (expressed sequence tag) the cDNA clone of PDE4A10 (BF528806), PDE4B2B (BC036108) and PDE4D2 (BF059733) is available from ATCC (American type culture collection (American Type Culture Collection)).The cDNA clone of people PDE4C2 is by University of Glasgow, and Miles professor Houslay of UK provides.The catalyst structure domain subclone of PDE4A10 (residue 298-622), PDE4B2B (residue 152-528), PDE4C2 (residue 200-558) and PDE4D2 (residue 79-438) is gone among the expression vector pET15b.Resulting plasmid pET-PDE4A, pET-PDE4B, pET-PDE4C and pET-PDE4D are transformed among the coli strain BL21 (CODONPLUS).Make the Bacillus coli cells that has described expression plasmid in the LB substratum, grow to photoabsorption A600=0.7 in 37 ℃, add 0.1mM isopropyl ss-D-sulfo-galactopyranoside then to induce expression 20-40 hour in 11 ℃-15 ℃.These recombinant proteins of PDE4A10, PDE4B, PDE4C2 and PDE4D2 carry out purifying on Ni-NTA avidity post (Qiagen), be zymoplasm cutting and being further purified on Q-SEPHAROSE (Amersham) and SUPERDEX 200 (Amersham) post subsequently.Typical purifying produces about 10mgPDE4A10,20mg PDE4B2B, 15mg PDE4C2 and 100mg PDE4D2 from 2 liters of cell cultures.These purified PDE4 protein demonstrate single strap in SDS-PAGE, have according to estimates＞95% purity.Scheme about the protein expression of PDE4D2 and purifying be disclosed (people such as Huai, 2003a).

The cDNA clone (U67733) of people PDE2A 3 is available from ATCC.Catalyst structure domain (residue 580-941) subclone of PDE2A3 is gone among the carrier pET15b, and in not containing the 2xYT substratum of NaCl, in coli strain BL21 (CODONPLUS), expressed 20 hours excessively in 15 ℃.Come the PDE2A3 of purification of Recombinant by Ni-NTA avidity, Q-SEPHAROSE and SEPHACRYL S300 post.Typical batch purifying produces about 5mg PDE2A3 from 2 liters of cell cultures.Described PDE2A3 albumen has＞95% purity and the specific activity of 180nmol/ minute/mg.

The cDNA clone of people PDE3A is provided by Robert doctor Colman of Temple University.The coding region subclone of the amino acid 679-1141 of PDE3A is gone among the pET15b.Plasmid pET-PDE3A crosses in coli strain BL21 (CODONPLUS) in salt-free modified LB substratum in 15 ℃ and expressed 24 hours.Come the PDE3A of purification of Recombinant by Ni-NTA avidity and SEPHACRYL S300 post.Typical purifying produces about 10mg PDE3A from 4 liters of cell cultures, purity is＞95%, and it has the specific activity of 120nmol/ minute/mg.

CDNA by ox PDE5A that Jackie doctor Corbin by Vanderbilt University is provided carries out site-directed mutagenesis and comes subclone people PDE5A1.With carrier pET15b and coli strain BL21 (CODONPLUS) be used in 15 ℃ of catalyst structure domains (residue 535-860) of crossing expressing human PDE5A1 spend the night (people such as Huai, 2004a).Come the PDE5A1 of purification of Recombinant by Ni-NTA avidity, Q-SEPHAROSE and SEPHACRYL S300 post.Typical batch purifying produces above 10mg PDE 5A1/2 and rises cell culture.PDE5A1 albumen has＞95% purity and have the specific activity of 2 μ M/ minutes/mg, and this and the total length PDE5A1 that in rhabdovirus system, expresses quite people such as (, 1999) Fink.

The cDNA of PDE7A1 is available from ATCC (BE782968).Catalyst structure domain (residue 130-482) subclone is gone among the carrier pET32a.Plasmid pET32-PDE7A1 is transformed among the coli strain BL21 (CODONPLUS), and in the 2xYT substratum, crosses expression 12-16 hour in 15 ℃.Use Ni-NTA avidity post, zymoplasm cutting subsequently and Q-SEPHAROSE and SEPHACRYL S300 post to come the catalyst structure domain of purifying PDE7A1.Typical purifying produces about 5mg PDE7A1 from 4 liters of cell cultures, it has according to estimates and is better than 95% purity people such as (, 2005) Wang.

The cDNA clone (BC009047) of people PDE9A2 is available from ATCC.Catalyst structure domain (residue 181-506) subclone of PDE9A2 is gone among the carrier pET15b, and in the LB substratum, in coli strain BL21 (CODONPLUS), cross expression in 15 ℃ and spend the night.Use Ni-NTA avidity, Q-SEPHAROSE and SEPHACRYL S300 post to come the PDE9A2 of purification of Recombinant.This PDE9A2 albumen has the purity greater than 95%, as by as shown in the SDS-PAGE.Typical purifying from 2 liters of cell cultures, produces above 100mg PDE9A2 (people such as Huai, 2004b).

The cDNA clone (BQ435652) of people PDE10A2 is available from ATCC.Catalyst structure domain (residue 442-789) subclone of PDE10A2 is gone among the carrier pET15b, and in not containing the 2xYT substratum of NaCl, in coli strain BL21 (CODONPLUS), cross expression 15-20 hour in 18 ℃.Come the PDE10A2 of purification of Recombinant by Ni-NTA avidity, Q-SEPHAROSE and SEPHACRYL S300 post.Typical purifying produces about 10mg PDE10A2 from 2 liters of cell cultures.This PDE10A2 albumen has＞95% purity, and as by as shown in the SDS-PAGE.

Use the PDE enzyme above discussed, assess described synthetic compound in conjunction with the ability of above-mentioned PDE family with regard to them.Based on for suppressing active assessment, the existence of some functionality and in core texture for finding to have strong dependency between the high binding affinity of some PDE4 subfamily and the selectivity.

Especially, four kinds of compounds (being presented in Fig. 2 and table 1 and 2) have the IC that is lower than micro-molar concentration (sub-micromolar) ₅₀Value.More importantly, these inhibitor of great majority demonstrate preference PDE4, and this shows to have excellent selectivity.For example, compound 1 has the IC of 18nM ₅₀, its selectivity is respectively 5000 and 27000 times for PDE7A1 and PDE9A2.

In another experiment, assess compound 1 with regard to its ability in conjunction with other PDE4 receptor subtypes.Compound 1 suppresses the IC of PDE4A, 4B, 4C and 4D ₅₀Value be respectively 99+/-15,35+/-5,127+/-17 and 18+/-1nM.

Therefore, to go out be selectivity PDE4 inhibitor to described compound exhibits.

The representative compounds of table 1. formula 7

The selected guide's inhibitor that goes out of table 2. is for the IC of various PDEs ₅₀Value (μ M)

Compound number	PDE4D2 (79-438)	PDE7A1 (130-482)	PDE9A2 (181-506)	His- PDE2?A3 (580-941)	PDE5A1 (535-860)
						1	0.018	100	500	100	500
2	35	360	150	1000	1000
						3	6	200	500	100	80
4	45	500	1000	1000	1000
						5	3.5	1000	1000	500	150
6	80	360	1000	1000	300
						7	10	36	50	100	20
8	6	17	60	300	40
						9	0.028	ND	ND	ND	ND
10	0.41	ND	ND	ND	ND
						11	0.061	ND	ND	ND	ND

The ND=undetermined

Embodiment 2: based on the PDE4 inhibitor assay method of cell

Be used to measure of the influence of described compound with following about program for tumor necrosis factor alpha (TNF α) generation aspect based on the PDE4 inhibitor assay method of cell.TNF α activates neutrophilic granulocyte and monocytic inflammatory cytokine, thereby plays an important role in chronic obstructive pulmonary disease (COPD) and asthma.Lipopolysaccharides (LPS) is crucial inflammatory stimulus thing, and it induces TNF α to produce and cause injury of lung.It is to be used for assessing the PDE4 inhibitor at the basic assay method of the effectiveness of anti-inflammatory and treatment COPD and asthma (people such as Muise, Biochem Pharmacol.63,1527-1535 (2002) that inhibition is produced by LPS inductive TNF α; People such as Ouaqued, Pulm.Pharmacol.Ther.18:49-54 (2005)).

Compound 1 and compound 9 have been measured in human peripheral blood mononuclear cell (PBMC) system in the activity that suppresses aspect LPS inductive TNF α release.The IC of compound 1, compound 9 and rolipram ₅₀Value is respectively 0.04,0.11 and 0.06 μ M (among Fig. 3 shown data).Since the variation of assay method system, the IC in the document about TNF α release ₅₀Value may not can directly compares.Yet, if with rolipram as relatively reference, the effectiveness of our compound 1 is than the much better (IC of rolipram of cilomilast ₅₀=1.3 μ M, the IC of cilomilast ₅₀＞10 μ M, people such as Oaqued, Pulm Pharmacol Ther.18,49-54 (2005)), cilomilast is the medicine lead that has been used for the treatment of COPD in clinical trial.

Foregoing is of the present invention illustrating, and is not interpreted as having limited the present invention.The present invention is defined by following claim, and the equivalent form of value of described claim is included in wherein.

Be listed in the reference of being quoted in the whole specification sheets down, be indicated as being relevant content in this article, integrate with this paper by mentioning with regard to it.

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Claims (20)

1. the compound of following formula:

Formula 1 formula 2 formulas 3

Wherein,

R ₁Be haloalkyl,

R ₂Be C _3-6The heterocyclic radical part of the cycloalkyl of cycloalkyl, replacement, heterocyclic radical or replacement comprises cyclopentyl, tetrahydrofuran base and oxa-cyclohexyl part especially,

R ₃Be selected from C _2-10Alkyl or haloalkyl, for example C _2-5Alkyl, more particularly C ₄Alkyl comprises normal-butyl, isobutyl-and the tertiary butyl,

R ₄And R ₅Be independently selected from C _2-6Alkyl, C _3-6Cycloalkyl, haloalkyl, perhaps both be combined to form carbonyl, imines (that is ,=NH) or the thiocarbonyl part,

X, X ' and X " are independently selected from N, C-H, C-F, C-Cl, C-Br, C-I, C-R ₆, C--NR ₆R ₇, C-CF ₃, C-CN, C-NO ₂, C-C ₂R ₆, C-SCH ₃, C--N ₃, C-SO ₂CH ₃, C-OR ₆, C-SR ₆, C-C (=O) NR ₆R ₇, C-NR ₆C (=O) R ₆, C-C (=O) R ₆, C-C (=O) OR ₆, C (CH ₂) _nOR ₆, C-OC (=O) R ₆, COC (=O) NR ₆R ₇And C-NR ₆C (=O) OR ₆, condition is X, X ' and X " in be no more than two for N,

Y is O, S or NH,

Wherein, R ₆And R ₇Hydrogen, C respectively do for oneself _2-6Alkyl, C _6-10Aryl ,-C _1-10Alkyl-C _6-10Aryl ,-C _6-10Aryl-C _1-10Alkyl, heteroaryl and

N is 1 to 10 integer.

2. the compound of claim 1, wherein R ₂Be selected from

Wherein Y as defined above.

3. the compound of claim 1, wherein R ₂Be selected from

4. the compound of claim 1, wherein R ₆Be

5. the compound of claim 1, it is defined by following formula:

Formula 4 formulas 5 formulas 6

Wherein, R _1-6, " and Y defines in claim 1 as mentioned for X, X ', X.

6. the compound of claim 1, it is defined by following formula:

Formula 7 formulas 8 formulas 9

Wherein, R _1-6In claim 1, define as mentioned with Y.

7. the compound of claim 1 or its pharmacy acceptable salt, wherein R ₁Be CHF ₂

8. the compound of claim 1 or its pharmacy acceptable salt, wherein R ₂Be tetrahydrofuran base or pyrrolidyl.

9. the compound of claim 1 or its pharmacy acceptable salt, wherein R ₂Be THP trtrahydropyranyl (amylene oxide).

10. the compound of claim 1 or its pharmacy acceptable salt, wherein R ₂It is cyclopentyl.

11. the compound of claim 1 or its pharmacy acceptable salt, wherein R ₄And R ₅Form carbonyl, thiocarbonyl or imines part together.

12. the compound of claim 1 or its pharmacy acceptable salt, wherein R ₃Be normal-butyl, isobutyl-, sec-butyl or the tertiary butyl.

13. pharmaceutical composition, it comprises among the claim 1-12 each compound and pharmaceutically acceptable carrier.

14. suppress the method for PDE-4 in Mammals, it comprises to each compound among the claim 1-12 of described administration treatment significant quantity.

15. having, the method for claim 14, wherein said Mammals be selected from following symptom: asthma, chronic bronchitis or chronic obstructive pulmonary disease.

16. having, the method for claim 14, wherein said Mammals be selected from following symptom: the septic shock of the Sepsis of endotoxin shock, septic shock, ulcerative colitis, bacterium or fungal induction, the Sepsis of virus induction, bacterium or fungal induction or the septic shock of virus induction.

17. having, the method for claim 14, wherein said Mammals be selected from following symptom: the reperfusion injury of Crohn's disease, cardiac muscle and brain, inflammatory arthritis, chronic glomerulonephritis, urticaria, rheumatoid arthritis, transplant rejection, graft versus host disease (GVH disease), inflammation mediated chronic tissue's sex change, cytokine mediated chronic tissue's sex change, osteoarthritis or amyotrophy.

18. having, the method for claim 14, wherein said Mammals be selected from following symptom: the cancerous invasion of the chronic obstructive pulmonary disease in adult respiratory distress syndrome, the animal, diabetes insipidus, allergic rhinitis, anaphylaxis conjunctivitis, vernal conjunctivitis, arterial restenosis, ortherosclerosis, atherosclerosis, neurogenic inflammation, pain, cough, ankylosing spondylitis, gastroxia, cancer, emaciation, dysthymia disorders, dysmnesia, tumor growth or healthy tissues.

19. having, the method for claim 14, wherein said Mammals be selected from following symptom: unipolar depression, acute and chronic neurodegenerative disorders, Parkinson's disease, mild cognitive impairment, the loss of memory, alzheimer's disease, spinal cord injuries receptor, craniocerebral injury or multiple sclerosis with inflammatory component.

20. having, the method for claim 14, wherein said Mammals be selected from following symptom: cardiovascular disorder or cancer.

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