CN1057772C - Compositions and methods for the treatment of immunomediated inflammatory disorders - Google Patents

Abstract

Compositions and methods for the prevention and treatment of immunoloregulation inflammatory disorders, especially for those disorders associated with the respiratory tract, are provided in the invention. More particularly, a tryptase inhibitor, typically a hydroxyaroyl or hydroxyheteroaroyl substituted dipeptide, is administered. Also provided by this invention are pharmaceutical compositions, typically aerosol or topical preparation, as well as aerosol devices for administering these compositions intranasally.

Description

Treat the compound and the composition of immunoregulatory inflammation

The application is the part continuation application of No. 08/031,187, copending application, and the application relates to the theme of No. 08/030,770, U.S. Patent application.These two applyings date in first to file are on March 12nd, 1993, and denomination of invention is respectively " composition and method for the treatment of immunoregulatory inflammation " and " treating the method for the strong responsiveness relevant with chronic asthma ".Clearly these copending applications are incorporated by reference at this paper.

The present invention relates generally to prevent and treat the composition and the method for immunoregulatory inflammation.More specifically, the present invention relates to prevent inflammation such as asthma and the allergic rhinitis relevant with respiratory tract with treatment.The segmental bronchus in late period that the compositions and methods of the invention are used in particular for preventing or treatment is relevant with chronic asthma dwindles and the strong responsiveness of air flue.

But have now found that inhibitory enzyme particularly the low-molecular weight compound of proteolytic enzyme can be widely used in the treatment various pathologic conditions.Many these compounds are called as peptide mimics, because as their name referring, these compounds have simulated the various inhibitor peptides of only being made up of condensation amino acid.These low molecular peptide mimic proteinase inhibitor are compared the special benefits that has with native peptides and are that they are more stable with protein, because they have required pharmacokinetic property and pharmacological properties.

For example, the zinc metalloprotein enzyme that is called Zinc metallopeptidase Zace1 (ACE) is determining angiotensin I to be split into Angiotensin II.Suppressing ACE is the hypertensive method of a kind of control.ACE can be suppressed effectively by the sulfydryl acyl derivative of proline(Pro), particularly Weller ﹠amp; Gordon is at United States Patent (USP) 4,456, disclosed dipeptide analogue (the 1-[(2S)-3-sulfydryl-2-methylpropionyl that is called captopril in 595]-L-proline(Pro) and analogue thereof.

Serine proteinases thrombin is a kind of key enzyme in the cascade that causes clot is solidified.By the medicine Trombin inhibiting is a kind of method that clot solidifies trend that reduces, so it has reduced and can cause having a heart attack or the thrombotic possibility of apoplexy.Zymoplasm can particularly be called arginic amino acid derivative by many peptide mimicses and surrogate suppresses effectively.People such as okamoto are at United States Patent (USP) 4,201, in 863 disclosed class N (2)-aryl sulfonyl-L-arginine amide particularly argipidine (arrgatroban) be this analoglike inhibitor.

Asthma is a kind of syndromes, comprises multiple biochemical medium for its acute and chronic performance.Asthma common is characterised in that for the specific allergen of immunity and the progressive development of strong responsiveness of general chemistry or material incentive trachea and bronchus.The strong responsiveness of the bronchiole tissue of asthma is considered to be caused by chronic inflammatory reaction, and inflammatory reaction stimulates and damage is gone up the airway walls in the leather sheet and impelled the pathologic thickening of lower-hierarchy.The segmental bronchus biopsy studies shows even the patient of mild asthma has the inflammation feature in airway walls.

Inflammatory process initial is the allergen allergic response to sucking.The white corpuscle that has an IgE acceptor is mastocyte and basophilic leukocyte but also comprise that monocyte, scavenger cell and eosinocyte are present in bronchial epithelium and the bottom smooth muscle tissue particularly, and begins to activate above-mentioned cell by antigen and the IgE receptors bind that makes specific suction.The activatory mastocyte discharges the chemical mediator and the enzyme of preformed or initial in a large number inflammatory response.In addition, a large amount of secondary inflammatory mediators generate by the enzyme reaction of activatory mastocyte is on-the-spot, comprise super-oxide and lipid deutero-medium.In addition, the threshing by mastocyte can discharge several macromole: protein-polysaccharide, peroxidase, ARB, particularly tryptase and Quimotrase (chymase).Referring to " Drug Thera-py of Asthma ", chap 62,1054-54.

This chemical release of mastocyte can explain that the early stage bronchiole constrictor that exists replys in the individuality of sensitivity after being exposed to airborne allergen.It is maximum that early stage asthma reaction reached after being exposed to allergen in about 15 minutes, then recovers in 1 to 2 hours.In the 25-35% individuality, then begin further decline behind the early stage asthma reaction, and after exposure, reached maximum in 6 to 12 hours several hours internal respiration functions.The later stage asthma reaction is followed and is penetrated into bronchiole unstriated muscle and epithelium and carefully knit and flow into the inflammatory cell quantity of air flue and obviously increase.These cells comprise eosinocyte, neutrophilic leukocyte and lymphocyte, and all these cells attracted to above-mentioned position by the release of mastocyte derivative reagent.Infiltrating cell itself in the late phase reaction stage is activated.Later stage asthma is replied and is considered to the secondary inflammatory reaction partly regulated by the secretion activity of scavenger cell.

A relevant class inflammatory reaction occurs in the upper respiratory tract mucosa, normally to airborne allergenic replying.When being in the asthma state, mastocyte is activated by IgE molecule and specific antigen cross-linking.For allergic perennial vasculomotor rhinitis, do not having discerniblely to be exposed to that mastocyte also can be activated under the specific antigen.In either case, the activatory mastocyte discharges the primary and secondary inflammatory mediator when threshing.Eosinocyte and scavenger cell attracted to above-mentioned position to keep inflammatory reaction.The destruction of nasion epithelium often occurs in the late phase reaction.

Class pancreas egg enzyme is the main extracellular proteinase of people's mastocyte, and is believed to comprise in neuropeptide processing and tissue inflammation.Grownup's tryptase is a kind of relevant tetramer of glycosylated and heparin of heterogeneous catalyst activatory subunit.There is not approaching corresponding thing in a large amount of other serine stretch protein acid of the same characterization of class Trypsin acid mono aminoacid sequence with its unit structure.Referring to as people such as Vander-slice (1990) Pron.Natl.Acad.Sci.USA 87:3811-3815; People such as Miller, (1990) J.Clin Invest.86:864-870; People such as Miller (1989) J.Clin.Inrest.84:1188-1195, people such as Vander-slice (1989) Biochemistry 28:4148-4155; With people (1990) Monographs in Allergy 27:51-66 such as Katunuma.

Tryptase is stored in the mastocyte secretory granule.After the mastocyte activation, in various biological liquids, be easy to measure human tryptase.For example, after anaphylaxis, tryptase appears in the blood flow, and keeps can surveying several hours.Referring to people such as Schwartz (1987) N.Engl J.Med.316:1622-1626.In the sample of the nasion that stimulates the atopy subject with specific antigen and lung-douching fluid, can measure the existence of tryptase.Referring to Castells﹠amp; People (1988) Am.Rev.Resp.Dis.141:563-568 such as Schwartz (1988) J.Allerg.Clin.Immunol.82:348-355 and Wenzel.Tryptase level in the lung-douching fluid that is obtained by the atopy asthma patient after the tunica mucosa bronchiorum allergen intensifies increases.Equally, the tryptase level of comparing some smokers' bronchoalveolar lavage fluid with the collator of non-smoking significantly improves, and this discovery impels the hypothesis of LD in smoker's wind-puff that support is provided to the release of the proteolytic enzyme of activation mastocyte.Referring to people such as Kalenderi-an (1988) (chest 94:119-123.In addition, proved that now tryptase is a kind of fibrocellular effective mitogen, thereby shown that it is relevant with a matter tuberculosis with pulmonary fibrosis.Referring to people such as Ruoss (1991) J.Clin.In-vest.88:493-499.

Tryptase is relevant with various bioprocesss, and the degraded that comprises the lax neuropeptide of vasorelaxation and segmental bronchus is (referring to people such as Caughey (1988) J.pharma-col.Exp.Ther.244:133-137; People such as Franconi (1988) J.Pharmacol.Exp.Ther.244:133-137; People such as Franconi (1988) J.Pharmacol.Exp.Ther.248:947-951; With people (1990) Am.J.Respir. (ell Mol.Biol.3:27-32) such as Tam with for the adjusting (referring to people such as Sekizawa (1989) J.Clin.Invest.83:175-179) of histamine segmental bronchus responsiveness.These studies show that tryptase may increase the contraction of asthma mesobronchus by destroying the bronchiectasis peptide.

In addition, verified tryptase can devillicate proteinogen α-chain and the high-molecular-weight kininogen that has releasable kassinin kinin, therefore can be with heparin as the agent of partially anti-freezing function blood.The class Trypsin shows that by the ability that MMP-3 activates prostromelysin (proMMP-3) and precollagen enzyme (pro-MMP-1) tryptase is also relevant with reconstruction with the inflammation of tissue.This discovery also shows tryptase generation effect in the destruction of joint of rheumatoid arthritis.Proved that in addition tryptase can divide the peptide relevant with Procalcitonin.Because this peptide and neuron inflammation-related, so tryptase is an important factor in the adjusting of the trigger reaction of the former inflammation of cutaneous nerve.Referring to Caughey (1991) Am.J.Respir.Cell Mol.Biol.4:387-394.Recently, reported that various forms of tryptases can be split into zymoplasm with prothrombin, and regulated the identification (referring to people such as Katununa (1990) Monographs inAllergy 27:51-66) of HIV virus membrane antigen gp120 by the CD4+T lymphocyte.

In industrialized country, asthma has become prevailing chronic disease.Up to now, Chang Gui method and therapeutical agent can not be treated asthma or other immunoregulatory inflammation effectively.Therefore be desirable to provide the shortcoming of avoiding these conventional formulations and method and can treat the improved composition and the method for these diseases simultaneously effectively.

The invention provides new tryptase inhibitors, it comprises on formula I compound or its medicine can accept to use salt, Wherein:

Ar is the aryl of hydroxyl replacement or the heteroaryl that hydroxyl replaces, and wherein hydroxyl is positioned at the ortho position of amide side chains, if Ar is the aryl that hydroxyl replaces, the aromatic ring of the amide side chains that has is not so replaced and do not have low alkyl group on the ortho position of hydroxyl by halogen;

R ¹Be hydrogen, low alkyl group, arylalkyl or heteroarylalkyl;

R ²Be hydrogen or low alkyl group;

R ³Be selected from:

Wherein m is the integer of 3-6, and n is the integer of 0-3, and p is the integer of 0-2, and q is the integer of 0-2; R is the integer of 0-5; S is the integer of 0-2; T is the integer of 1-3; U is 1 or 2; V is the integer of 3-6; W is the integer of 0-3; A is-CH=CH-or-C ≡ C-; X is-NH-or-CH ₂-;

R ⁴Be low alkyl group, the arylalkyl of replacement or the heteroarylalkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroarylalkyl of replacement; Or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen; Or R ⁴And R ⁶With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁵Be hydrogen;

R ⁷For-OR ⁸Or-NR ⁸R ⁹, R wherein ⁸And R ⁹Independently be selected from hydrogen, low alkyl group, aryl, arylalkyl or heteroarylalkyl, or R ⁸And R ⁹Form 5 yuan and 6 yuan of heterocycles of replacement with the nitrogen that they connected.

In preferred embodiments, Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridyl or 2-hydroxyl-3-quinoxalinyl; R ¹Be hydrogen; R ²Be hydrogen; R ³Be selected from:

Wherein m is the integer of 3-6, and n is the integer of 0-3, and p is the integer of 0-2, and q is the integer of 0-2; W is the integer of 0-3; A is-CH=CH-or-C ≡ C-; X is-NH-or-CH ₂-;

R ⁴Be low alkyl group, R ⁵And R ⁶Be hydrogen; Or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen; R ⁷For-OH ,-OCH ₃,-NH ₂, 3 '-hydroxy amino-1 '-piperidyl or-N (CH ₃) ₂

The tryptase inhibitors of particularly preferred formula I is the compound 3 of Table I and II:

The tryptase inhibitors of another preferred formula I is the compound 15 of Table I and II:

Compound as herein described can be used for prevention and treats immunoregulatory inflammation, and particularly relevant with respiratory tract inflammation comprises asthma and particularly relevant strong responsiveness phase and the rhinallergosis with chronic asthma.Therefore, the present invention also provides the method for the treatment of immunoregulatory inflammation, wherein suffers from the patient with the responsive immunoregulatory inflammation of tryptase inhibitors treatment is accepted or take treatment effective dose or a certain amount of The compounds of this invention.

The present invention also provides the pharmaceutical composition of compound described herein.These pharmaceutical compositions have various formulations and comprise oral dosage form and injectable and pourable solution.Usually, when being used for the treatment of or during strong responsiveness that prevention of asthma is particularly relevant with chronic asthma, these pharmaceutical compositions can adopt the aerosol of pulvis or solution.When being used for the treatment of immunoregulatory inflammation tetter, The compounds of this invention can be used in combination with nontoxic pharmaceutically useful topical carrier.Compound of the present invention can be used in combination with the reflunomide of antiphlogistic drug or other treating asthma medicines such as beta-adrenergic antagonistic, anti-inflammatory, anticholinergic etc.

Fig. 1 for the specific lung resistance of expression sheep as antigenic stimulation after the time (hour) the curve of function.Open squares represents to contrast class value, and the value of identical animal behind the compound 3 of taking Table I and II represented in hollow garden.

Fig. 2 be illustrated in take long crust can before the strong responsiveness generation of the sheep figure of 24 hours derivable bronchoconstriction of long crust after the antigenic stimulation take Table I and II compound 2 time.The solid black bar is corresponding to control group, benchmark.Light color solid bars corresponding to medicine, benchmark.The shade lines are corresponding to control group, behind the antigen.White bars is corresponding to medicine, behind the antigen.

I. define and general parameter

Following definitions is used for explanation and defines implication and the scope that is used for describing various terms of the present invention.

" immunoregulatory inflammation " generally comprises and discharges relevant with labrocyte medium and to the responsive disease of tryptase inhibitors treatment. The example of these diseases comprises immediate hypersensitivity disease such as asthma, allergic rhinitis, nettle rash, angioedema, eczematous dermatitis (atopic dermatitis), allergic reaction, with anti-high proliferative skin disorders, peptic ulcer, enteritis, dermatitis etc.

" strong responsiveness " refers to later stage bronchoconstriction and the airway hyperreactivity relevant with chronic asthma. The strong responsiveness of the bronchiole tissue of asthma is considered to be caused by chronic inflammatory reaction, inflammatory reaction stimulate and the upper leather sheet of operation in airway walls and impel the pathologic thickening of lower-hierarchy.

" halogen " refers to fluorine, bromine, chlorine and iodine atom.

" hydroxyl " refers to group-OH.

" low alkyl group " refers to ring-type, the branched-chain or straight-chain alkyl of 1 to 6 carbon atom. The group that this term can further be enumerated is methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group (or 2-methyl-propyl), cyclopropyl methyl, n-pentyl and hexyl.

" aryl " or " Ar " refer to have monocycle (such as phenyl), many rings (such as xenyl) or wherein at least one ring be that many fused rings of aromatic ring are (such as 1,2,3,4-tetralyl, naphthyl, anthryl or phenanthryl) aromatic carbocyclyl groups, it can be chosen wantonly is not replace or replaced by following groups: halogen, low alkyl group, lower alkoxy, lower alkylthio, trifluoromethyl, low-grade acyloxy, aryl, heteroaryl and hydroxyl. Yet, according to the present invention, can not further be replaced by halogen with the aromatic ring of amide side chains. In addition, the ortho position of hydroxyl (be amide side chains between position) low alkyl group can not be arranged with the aromatic ring of amide side chains.

" heterocycle " refer to have monocycle (for example morpholino, pyridine radicals or furyl) or many fused rings (for example 1.5 1 phthalazinyls, quinoxalinyl, quinolyl, indolizine base or benzo [b] thienyl) and have saturated, the unsaturated or aromatic carbocyclyl groups of at least one hetero atom such as N, O or S in ring, it can be chosen wantonly is unsubstituted or is replaced by following groups: halogen, low alkyl group, lower alkoxy, lower alkylthio, trifluoromethyl, low-grade acyloxy and hydroxyl. Term " heteroaryl " and " Het-Ar " refer to that wherein at least one heterocycle is the heterocycle of aromatic ring.

" aralkyl " refer to group-R-Ar wherein Ar be that aryl and R are the straight or branched aliphatic group. Aralkyl can be chosen wantonly to be unsubstituted or to be replaced by following groups: halogen, low alkyl group, lower alkoxy, lower alkylthio, trifluoromethyl, low-grade acyloxy and hydroxyl.

" heteroaryl alkyl " refer to group-R-HetAr wherein HetAr be heteroaryl, R is the straight or branched aliphatic group. Heteroaryl alkyl can be chosen wantonly to be unsubstituted or to be replaced by following groups: halogen, low alkyl group, lower alkoxy, lower alkylthio, trifluoromethyl, low-grade acyloxy and hydroxyl.

" pharmaceutically acceptable salt " refers to keep biological effect and the character of parent compound, and is not biologically or other unwanted salt.

" medicine or treatment upper acceptable carrier " refers to not affect the biologically active effect of active ingredient and to host or the nontoxic mounting medium of patient.

" stereoisomer " refers to have same molecular amount, chemical composition and structure, but atom is arranged different compounds each other. That is to say that some identical chemical part is in different orientations in the space, therefore, pure stereoisomer can the rotatory polarization optical plane. Yet the optical activity that some pure stereoisomer has is very low and can not measure with modern instrument. Compound of the present invention has one or more asymmetric carbon atoms, therefore comprises various stereoisomers. All stereoisomers all are included among the scope of the present invention.

" treatment " refer to external or body in use tryptase inhibitors, comprising:

(i) symptom of inhibition disease;

(ii) alleviate or suppress the long term of disease; And/or

The symptom that (iii) palliates a disease.

II. tryptase inhibitors

The invention provides and comprise effective serpin, more particularly is the composition of tryptase inhibitors, it can be used for alleviating immunoregulatory inflammation particularly the asthma animal stimulate the inductive bronchoconstriction by allergen.

Tryptase inhibitors is to reduce or the active material of prevention tryptase.According to a further aspect in the invention,

Tryptase inhibitors comprises formula I compound or its pharmaceutically acceptable salt:

Ar is the aryl of hydroxyl replacement or the heteroaryl that hydroxyl replaces, and wherein hydroxyl is positioned at the ortho position of amide side chains, if Ar is the aryl that hydroxyl replaces, the aromatic ring that has amide side chains is not so replaced and do not have low alkyl group on the ortho position of hydroxyl by halogen;

R ¹Be hydrogen, low alkyl group, arylalkyl or heteroarylalkyl;

R ²Be hydrogen or low alkyl group;

R ³Be selected from:

Wherein m is that the integer of 3-6: n is the integer of 0-3; P is the integer of 0-2; Q is the integer of 0-2; R is the integer of 0-5; S is the integer of 0-2; T is the integer of 1-3; U is 1 or 2; V is the integer of 3-6; W is the integer of 0-3; A is-CH=CH-or-C ≡ C-; X is-NH-or-CH ₂-;

R ⁴Be low alkyl group, the arylalkyl of replacement or the heteroarylalkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroarylalkyl of replacement; Or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen; Or R ⁴And R ⁶With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁵Be hydrogen;

R ⁷For-OR ⁸Or-NR ⁸R ⁹, R wherein ⁸And R ⁹Independently be selected from hydrogen, low alkyl group, aryl, arylalkyl or heteroarylalkyl, or R ⁸And R ⁹Form 5 yuan or 6 yuan of heterocycles of replacement with the nitrogen that they connected.

In preferred embodiments, Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridyl or 2-hydroxyl-3-quinoxalinyl; R ¹Be hydrogen; R ²Be hydrogen; R ³Be selected from:

Wherein m is the integer of 3-6, and n is the integer of 0-3, and P is the integer of 0-2, and q is the integer of 0-2; W is the integer of 0-3; A is-CH=CH-or-C=C-; X is-NH-or-CH ₂-;

R ⁴Be low alkyl group, R ⁵And R ⁶Be hydrogen, or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen; R ⁷For-OH ,-OCH ₃,-NH ₂, 3 '-amino carboxyl-1 '-piperidyl or-N (CH ₃) ₂

Preferred tryptase inhibitors is the compound 3 of Table I and II:

The compound 15 that another preferred tryptase inhibitors is Table I and II:

The compound 21 that another preferred tryptase inhibitors is Table I and II:

Tryptase inhibitors of the present invention can be made by the starting raw material that is easy to obtain with currently known methods, hereinafter will describe in more detail.

The character that depends on functional group, The compounds of this invention can form additive salt with various inorganic and organic bronsted lowry acids and bases bronsted lowries.Form mineral acid example hydrochloric acid, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid of these salt etc., organic acid such as acetate, propionic acid, oxyacetic acid, pyruvic acid, oxalic acid, oxysuccinic acid, propanedioic acid, succsinic acid, toxilic acid, fumaric acid, tartrate, citric acid, phenylformic acid, styracin, amygdalic acid, methylsulfonic acid, ethyl sulfonic acid, tosic acid, Whitfield's ointment etc.

Also can pass through with basic metal or alkali metal base such as alkali metal hydroxide and alkali metal alcoholates or alkaline-earth metal or alkaline earth metal alkali such as alkaline earth metal hydroxides and alkaline-earth alkoxides processing hydroxy-acid group generation salt.In addition, also can generate salt, organic bases such as Trimethylamine 99, diethylamine, thanomin, piperidines, Isopropylamine, choline, caffeine etc. by carboxylic acid and organic bases.

These salt can prepare with ordinary method, suitable alkali of the free acid of product or alkali form and monovalent or a few equivalent or acid are reacted in insoluble solvent of salt or medium or under can vacuum or in solvent of removing by lyophilize such as the water, or on suitable ion exchange resin, make the cationic exchange of existing salt be other positively charged ion.

III. external and in vivo test

The in vitro tests method of screening effective inhibitors according to the ability that suppresses tryptase is known in the prior art.For example referring to people such as Sturzebecher (1992) Biol.Chem.Hoppe-Seyler 373:1025-1030.Usually, the hydrolysis of these experimental measurement tryptase inductive peptidyl chromogen substances.The details of concrete grammar will be described below.

In addition, one of active available a large amount of animal model in asthma of The compounds of this invention are carried out in vivo test and are assessed.Referring to Larson, " Experimental Modelsof Reversible Airway obstruction ", The Lung:ScientificFoundations, Crystal, people such as West, eds., Raven Press, New York, 1991; People such as Warner (1990) Am.Rev.Respir.Dis.141:253-257.Ideal animal model should be able to duplicator's asthma main clinical and physiological characteristic, comprise: to the strong responsiveness of air flue of chemical mediator and physical stimulation, the medicine (β-adrenergic, methyl xanthine, reflunomide etc.) that is used for people's asthma can make obstruction of the air passage reverse; The leukocytic infiltration of activatory produces airway inflammation; The sex change of chronic inflammatory diseases changes, for example basement membrane thickening, unstriated muscle hypertrophy and epithelial damage.The kind that can be used as animal model comprises mouse, mouse, cavy, rabbit, dog and sheep.All animals all have some restriction, but select animal model to depend on related problem suitably.

With cavy and dog particularly the initial asthma of basenji-greyhounol Hybrid assessment reply, it produces the strong responsiveness of unspecific air flue for a large amount of non-allergenic substances such as methacholine and citric acid.Exciting some selected sheep of back to show dual replying with the Ascaris proteantigen.Reply in the animal dual, after exposure 6-8 hour, initial asthma was replied (IAR) back and is produced later stage asthma and reply (LAR).Intensifying the back 24 hours hypersusceptibilities to the cholinergic agonist carbachol for those animals of performance LAR at antigen increases.

Assess the antasthmatic useful effect of The compounds of this invention with the allergy model.The composition of using the aerosolization solution that contains The compounds of this invention for before or after being exposed to specific allergen the allergy sheep proves that these compositions can alleviate greatly or get rid of that later stage asthma is replied and strong responsiveness subsequently.

Compound of the present invention also can be used for treating other immunoregulatory inflammation that tryptase activity wherein causes pathological state.These diseases comprise the inflammation relevant with mastocyte such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, urarthritis and other arthritis disease, enteritis, peptic ulcer and various tetter.

The compounds of this invention is used for the treatment of the effect of most of immunoregulatory inflammation and can assesses with external or In vivo assay Cells.Therefore, the antiphlogistic effects of The compounds of this invention can be determined with test well known in the art, for example reverse passive Ah figure's phase and react (RPAR)-PAW method (referring to people such as Ganguly (1992) U.S.5,126,352).Determine that it is known in the art that The compounds of this invention is treated the test of the therapeutic action of various tetter such as high proliferative skin disorders, for example peanut device olefin(e) acid mouse ear test (id).Assess the antiulcer activity of The compounds of this invention according to the described method of people such as Chiu (1984) Archives Interna-tion-ales de Pharmacodynamie et de Therapie 270:128-140.

IV. vivo medicine-feeding

According to the present invention, give particularly formula I compound of tryptase inhibitors that the patient suffer from immunoregulatory inflammation takes treatment or medicine effective quantity.According to a specific embodiments, composition of the present invention can be used for prevention or improves asthma.When using present composition treatment asthma, before being exposed to allergen or other burst factor or after so exposing, prophylactically take The compounds of this invention.The compounds of this invention is used in particular for improving visible later stage disorganization in seasonal and perennial rhinitis.Another aspect of the present invention relates to prevention other immunoregulatory inflammation relevant with mastocyte with treatment such as urticaria and angioedema, warm rash dermatitis (atopic dermatitis), allergy and high proliferative skin disorders, peptic ulcer etc.

The composition that contains The compounds of this invention can preventative and/or curative form administration.In treatment is used, take the composition of curing or suppressing disease symptoms and complication significant quantity thereof to small part for the patient who has suffered from above-mentioned disease.The consumption that is enough to achieve the above object is defined as " treatment significant quantity or dosage ".Significant quantity used herein depends on the severity of disease and stadium, former treatment, patient's healthy state, to the reaction of medicine and attending doctor's diagnosis.

In prophylactic applications, give responsive or be in insensitive patient of special disease critical days and take the composition that contains The compounds of this invention.So consumption is defined as " prevention significant quantity or dosage ", and in this used, accurate consumption also depended on patient's healthy state, body weight etc.

In case patient's situation improves, if necessary, take maintenance dose.Then reduce the dosage taken or frequency or both level to the situation that can keep improvement according to symptom.When being relieved to required degree, symptom then can stop treatment.Yet based on the recurrence of illness, patient needs long-term intermittent therapy.

Usually, the suitable effective dose of tryptase inhibitors is 0.1 to 1000 milligram (mg)/acceptor/sky, preferred 1 to 100mg/ day.Required dosage preferably with one, two, three, four or more a plurality of sub-doses form exist, in one day, connect suitable interval and take sub-doses.These sub-doses can be taken by unit dosage.For example the per unit formulation contains 5 to 1000mg preferred 10 to 100mg active ingredients.

Compositions for use can adopt various formulations in these treatments.They comprise solid, semisolid and liquid dosage form, as tablet, pill, pulvis, liquor or suspension, liposome, injection liquid and perfusion liquid.Preferred formulation depends on predetermined administering mode and treatment application.

Although can take active ingredient of the present invention separately, preferably the part as pharmaceutical preparation exists.Preparation of the present invention comprises at least a treatment or the The compounds of this invention of significant quantity or inhibitor and one or more the pharmaceutically useful carrier of usefulness and other optional treatment compositions of maybe can treating on the medicine.Various considerations have been described, for example at people such as Gilman (eds) (1990) Goodman﹠amp; Gilman ' s:The Pharmacological Bases of Therapeutics, 8thEd. is among Pergamon Press and the Remington ' s Supra.Discussed in the document that medication is for example oral, intravenously, intraperitoneal or intramuscular administration and other method.Pharmaceutically acceptable carrier comprises water, salt solution, damping fluid and at the MerckIndex, Merck ﹠amp; Co.Rahway, other compound of describing among the NJ..

Usually, when The compounds of this invention is used for the treatment of asthma or rhinallergosis, they are mixed with aerosol.Term " aerosol " comprise The compounds of this invention the suspension medium that can produce gas, it can be inhaled into bronchiole or nasal passage.Especially, aerosol comprises the droplets suspended liquid that can produce gas of The compounds of this invention, and they are prepared in metered dose inhaler or the atomizer.Aerosol also comprises the dry powder composition that is suspended in the The compounds of this invention in air or other vector gas, combines by inhalation from sucker.

For the solution that is used to prepare aerosol of the present invention, the concentration range of preferred The compounds of this invention is 0.1-1006 milligram (mg)/milliliter (mL), is 0.1-30mg/mL more preferably, most preferably is 1-10mg/mL.Solution is usually with the damping fluid that allows on the physiology such as phosphoric acid salt or buffered with bicarbonate.Common pH scope is 5 to 9, and is preferred 6.5 to 7.8, more preferably 7.0 to 7.6.The general sodium-chlor that adds is adjusted to biological scope with perviousness, preferably in 10% isotonicity.For the preparation that produces these solution that aerosol sucks has come into question in Remington ' sPharmaceutical Sciences, also can be referring to Ganderton ﹠amp; Jones, Drug Delivery to the Respiratory Tract, Ellis Hor-wood (1987); Gonda (1990) Critical Reviews in Thera-peutic Drug Carrier Systems 6:273-313; With people (1992) J.pharmacol.Toxicol.Methods 27:143-159 such as Raeburn.

Be converted into aerosol with being generally used for preparing known method that aerosol sucks medicament solution with The compounds of this invention.Usually, these methods comprise usually with the inert carrier gas pressurization or a kind of container pressurizing device that makes solution are provided, and the gas that makes pressurization is by the tubule mouth, thereby the droplet of solution is pushed in the mouth and tracheae of the animal that need take medicine.Adapter generally is positioned in the outlet of the mouth of pipe, so that be administered into easily in mouth and the tracheae.

In one embodiment, the inventive system comprises the solution that makes The compounds of this invention links to each other with the conventional equipment that is used to produce aerosol in asthma therapies or is loaded on wherein conventional equipment such as metered dose inhaler, jet nebulizer or ultrasonic nebulizer.These devices are chosen wantonly and are comprised the adapter that is positioned on the mouth of pipe.

In being used for the treatment of the embodiment of rhinallergosis, the inventive system comprises the solution of The compounds of this invention is loaded in the jetmizer.

The dry powder doses of the vehicle that contains The compounds of this invention and choose wantonly is another embodiment of the present invention.It can be by containing the drug powder sucker administration of above-mentioned pulvis.

Certainly, method of the present invention can with the immunoregulatory inflammation of treatment particularly other medicament of asthma be used in combination.β-melt adrenergic agonist can be used in particular for these in conjunction with in reply symptom because it can alleviate initial asthma, and The compounds of this invention can alleviate later stage asthma and replys.Preferred β-class parathyrine can comprise the β-agonist any commonly used that is used to alleviate asthma by agonist in these solution, as salbutamol, terbutaline, good fortune make a mistake alcohol (formoterol), fenoterol (fan-oterol) or prenaline.

Other medicament that is used in combination with The compounds of this invention comprises that the scorching sterol of cortex (adrenocortical steroid) of anticholinergic drug such as the anti-inflammatory of ipratropium bromide group is as beclomethasone, triamcinolone, flurisolide or dexamethasone.

Compound of the present invention also can be used for treating mammiferous immunoregulatory inflammation tetter such as urticaria and angioedema, eczematoid dermatitis and high proliferative skin disorders such as psoriasis.The topical of through type I compound is expected mitigation symptoms.Therefore suffering from the dermopathic patient of immunoregulatory inflammation is expected to alleviate scale, erythema, patch size, itch and other symptom relevant with tetter.Successfully treating the required drug dose of each individual patient is different with the time, but those skilled in the art should be able to recognize that these are different and can regulate according to therapeutic process.

The present invention also comprises the preparation that local skin is used, and it comprises that the general concentration of formula I compound is 0.0001% to 10% and the carrier of nontoxic pharmaceutically useful local usefulness.These topical formulations can prepare by conventional medicine thinner commonly used in the dried agent in active ingredient of the present invention and part, liquor, emulsifiable concentrate and the aerosol and carrier are mixed.For example, ointment and emulsifiable concentrate can and add suitable thickening and/or gelifying agent is prepared with water base or oil base.These matrix comprise that water and/or oil are as liquid mountain tallow or vegetables oil such as peanut oil or Semen Ricini oil.Character available thickening material according to matrix comprises sunshine, aluminum stearate, hexadecyl Stearyl alcohol, propylene glycol, polyoxyethylene glycol, lanolin, hydrogenant lanolin, beeswax etc.

Washing lotion can be prepared with aqueous or oily matrix, and it generally also comprises one or more following compositions: stablizer, emulsifying agent, dispersion agent, suspension agent, thickening material, tinting material, spices etc.

Pulvis can be by means of preparations such as any suitable pulvis matrix such as talcum, lactose, starch.Drops can be prepared with hydrated matrix or non-aqueous matrix, and it also can comprise one or more dispersion agents, suspension agent, solubilizing agent etc.

Local medicine composition of the present invention also comprises one or more sanitass or bacteriostatic agent for example methyl hydroxybenzoate, nipasol, parachlorometacresol, benzalkonium chloride etc.Local medicine composition also can contain other live bosom composition such as biocide particularly antibiotic, narcotic, anodyne and antipruritic.

Compound of the present invention also can be used for treating peptic ulcer.More specifically, they show the chemotherapy activity, and its activity makes them can alleviate the symptom of peptic ulcer disease and suppresses ulceration, and impels the healing of stomach and/or 12 batches of intestinal ulcer.The compounds of this invention can be used in combination with other therapeutical agent, for example antiphlogistic drug and/or anodyne such as Asprin, INDOMETHACIN, BUTE, ibuprofen, naprosine, Tolectin etc.

Can pass through parenteral or oral administration for preventing and/or treating pharmaceutical composition of the present invention.Depend on medication, pharmaceutical composition of the present invention can be with various unit dosage administrations.For example, be suitable for oral unit dosage and comprise pulvis, tablet, pill, capsule and dragee.

Pharmaceutical composition of the present invention can intravenous administration.Therefore, the invention provides the intravenous administration composition, it comprises and is dissolved in or is suspended in compound and the solution that can accept in the preferred aqueous carrier of carrier.Can use various aqueous carriers for example water, buffered water, 0.4% salt solution etc.The known sterilising method of the available sometimes routine of these compositions is sterilized or is carried out sterile filtration.The aqueous solution of gained is comprised use, for example carry out freeze-drying, before using, freeze dried preparation is mixed with aseptic aqueous solution, for reaching proximate physiological condition, these compositions can contain pharmaceutically useful auxiliary substance such as PH adjusting and buffer reagent, strong shape conditioning agent, gentle dose etc., for example sodium acetate, Sodium.alpha.-hydroxypropionate, sodium-chlor, Repone K, calcium chloride, sorbitan monolaurate, trolamine oleyl alcohol salt etc.

For solids composition, can use conventional non-toxic solid carrier, it comprises N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, talcum, Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate of pharmaceutical grade etc.For oral administration, pharmaceutically acceptable non-toxic composite can prepare by adding any vehicle commonly used, those listed carriers of front for example, and contain the 0.1-95% active ingredient usually, preferably approximately 20%.

For invention described herein is fully understood, list the following example.Certainly these embodiment only are used for explanation, and do not constitute any limitation of the invention.

Experiment I. general introduction

The starting raw material that this paper does not describe can buy from the market, be known maybe can be by method preparation well known in the art.Formula I compound can be combined to the technology preparation with solid or solution by method well known in the art.The starting raw material of preparation I compound be in the art known maybe can be by well known to a person skilled in the art method preparation.For example the solid phase synthesis technique of polypeptide is described in Solid Phase Pep-tide Synthesis:A Practical Approach, (eds.E.Atherton ﹠amp; R.C.sheppard) IRL Press at Oxford Unirersity Press (1989) and Merrifield are among the J.Amer.Chem.Soc.85:2149-2156 (1963).Peptide coupling chemistry also is described in The Peptides, Vol.1, (eds.Gross, E. ， ﹠amp; J.Meienhofer).Among the Academic Press Orlan-do (1979).Other technology comprises people such as Geysen, the method described in Nature (1991) 354:84-86.

In the method for preparing The compounds of this invention described herein, the group that needs protection is that the organic chemistry filed technician is known.Therefore, method as herein described must contain uses suitable protecting group, although do not offer some clarification on.

If necessary, available any suitable isolated or purified method is carried out separating and purifying of compound as herein described and intermediate.For example filter, extraction, crystallization, post look all, the combination of thin-layer chromatography or thick layer chromatography, high pressure liquid chromatography or these methods.Specifying of suitable separation and isolation process referring to the following examples.Certainly also can use the separation or the isolation process of other equivalence.

The Sakaguchi test is used to measure arginine and contains arginic peptide, referring to Stewart ﹠amp; Young " Solid Phase Peptide Synthesis ", 2d.Ed., Pierce Chemical Company, P.114.Preparation contains 95% ethanolic soln I of 0.01% naphthyl alcohol and 5% urea.The 2g bromine is dissolved in the 100ml8% aqueous sodium hydroxide solution prepares solution II.In solution I, add 5 sodium hydroxide.With sample point to be analyzed on thin-layer chromatography (TLC) plate.Launch the TLC plate with solution I then.The TLC plate at air drying, is launched with solution II then.Red point shows has amino imino methane group to exist.II. the solid phase synthesis of formula I compound

A. the preparation of the compound 3 of Table I and II

With 4-(2 ', 4 ' one Dimethoxyphenyl fluorenylmethyloxycarbonyl (Fmoc)-amino methyls) phenoxy resin (Rink resin; Bachem, CA; 3.5g, carry 0.289 milliequivalent/gram (meq/g) 1.01mmol altogether) and the be suspended in 30ml N of 1: 1 (v/v), N dimethyl formamide (DMF): contain in the solution of toluene of 30% (volume) piperidines.Reaction mixture was stirred 5 minutes, filter then.Add piperidine solution (30ml) again, and then mixture was stirred 5 minutes.After the filtration, use DMF (6x) and methylene dichloride (6x) washing resin particle successively.

With Fmoc-L-proline(Pro) (Milligen; 6.06mmol), benzotriazole-1-base-oxygen-three (dimethylamino) hexafluorophosphate (BOP, Nov-abiochem; 6.06mmol) and I-hydroxybenzotriazole (HOBT; Aldrich; 6.06mmol) be dissolved among about 30mlDMF.Settled solution with gained is added in the Rink resin then, and pulpous state liquid was stirred (using nitrogen bubble) 4 hours.Filter reaction mixture, and washing resin particle as stated above.Remove the Fmoc group with piperidines as stated above.

With Fmoc-L-arginine (PMC) (Milligen; 6.06mmol), DMF (30ml) solution of BOP (6.06mmol) and HOBT (6.06mmol) is added in the resin particle, then pulpous state liquid is stirred 4 hours, filters as stated above then, washs, handles and washing once more with piperidines.

Add 1-hydroxyl-2-naphthoic acid (Aldrich; 1.14g, 6.06mmol), 1,3-di-isopropyl carbodiimide (DIPCDI; Aldrich; 0.949ml, 6.06mmol) and HOBT (0.819g, DMF 6.06ml) (45mL) solution stirred (mechanical stirring bar) 7 hours with reaction mixture.Filter, washing and handle (as stated above) with piperidines after, use DMF (6x), methylene dichloride (6x) and methyl alcohol (6x) washing resin successively.Resin is suspended in the methyl alcohol, and stirs (mechanical stirring bar) spend the night (15 hours).Resin filter is also used twice of methanol wash.

Then with the free-pouring solid suspension of white in trifluoroacetic acid (TFA): methyl-phenoxide: water (90: 5: 5,30ml) solution.Resin becomes pink at once, and color change melt into is scarlet in 1-2 minute then.Stir after 5 minutes filter reaction mixture.Repeating above-mentioned steps 2 times with new division reagent, is each multiple reaction times to extend to 10 minutes.

Merge TFA solution, the clear orange solution of gained was at room temperature placed 3.5 hours.Vacuum concentration obtains light yellow oil, and keeps 3 hours down in vacuum (＜1 torr).Oily matter is dissolved in the crude product (471mg) that the fine aqueous solution of 50% second (100ml) and lyophilize obtain pale solid.

HPLC analyze (Polymer Labs 100A PLRP post, 1.0 * 150mm was with 20 to 45% acetonitrile linear gradient elutions 13 minutes, flow velocity 0.1mL/min), show that crude product is simplification compound (retention time is 10.35 minutes, and UV absorbs＞98%) substantially, by HPLC C ₁₈Silicagel column (Vy-dac; 22 * 250mm, 15-20 μ m, 300A,, flow velocity 10mL/min with 20 to 35% acetonitrile linear gradient elutions 30 minutes) carry out purifying.Need multiple injection 30-35mg, merge same cut (retention time is about 44-48 minute) and lyophilize and obtain loose white solid (351mg), analyzing through HPLC is the simplification compound.

EFI mass spectrum (molecular weight of calculating=440.49, measured value M ^{+ 1}=441.1) and NMR spectrum (proton and ¹³C) structure with expection is consistent.

H NMR (CD ₃OD) ﹠amp; 7.89 (d, J=8Hz, 1H), 7.78 (d, J=6Hz, 1H), 7.76 (d, J=6Hz, 1H), 7.57 (dt, J=8,1Hz, 1H), 7.48 (dt, J=8,1Hz, 1H), 7.29 (1H) ,-4.95 is (fuzzy for d, J=8Hz, 1H), 4.49 (dd, J=8,5Hz, 1H), 3.97 (dt, J=10,7Hz, 1H), 3.73 (dt, J=10,7Hz, 1H), 3.23 (t, J=7Hz, 2H), 2.20 (m, 1H), 1.7-2.2 (m, 8H)

B. the preparation of other formula I compound

According to the method for above-mentioned part A, replace 1-hydroxyl-2-naphthoic acid with following compounds:

2-hydroxy-4-methyl phenylformic acid;

3-hydroxyl-2-quinoxaline formic acid;

3-hydroxyl-2-naphthoic acid;

2-hydroxyl-1-naphthoic acid;

3-hydroxyl-2-pyridine carboxylic acid;

4-hydroxyl-7-methyl-3-(1,5 one naphthyridine) formic acid;

2-hydroxyl-3-pyridine carboxylic acid;

2 hydroxybenzoic acid;

2, the 5-resorcylic acid; With

2-hydroxyl-5-Phenylbenzoic acid (referring to following E) makes following compounds.

The compound 2 of Table I and II;

The compound 9 of Table I and II;

The compound 14 of Table I and II;

The compound 15 of Table I and II;

The compound 16 of Table I and II;

The compound 17 of Table I and II;

The compound 18 of Table I and II;

The compound 19 of Table I and II;

The compound 20 of Table I and II; And

The compound 21 of Table I and II;

C. the preparation of other formula I compound

Method according to above-mentioned part A.Replace the Fmoc-L-proline(Pro) with the Fmoc-D-proline(Pro).Obtain the bonded 12 of Table III.

D. the preparation of other formula I compound

The preparation of the compound 13 of Table I and II is the methods that are used for coupling Fmoc-L-proline(Pro) by being similar in above-mentioned part A, at first make Fmoc-L-phenylalanine and resin coupling, remove the Fmoc group then, make phenylalanine and the coupling of Fmoc-L-proline(Pro), finish compound 13 synthetic rest parts with the described method of above-mentioned part A.

Equally, the compound 6 of Table I and II and 7 preparation comprise make Fmoc-piperidines-3-formic acid or Fmoc-piperazine shallow lake-4-formic acid respectively with the resin coupling, remove the Fmoc group, with the coupling of Fmoc-L-proline(Pro), finish the synthetic rest part then with the described method of above-mentioned part A.

E.2-hydroxyl-5-phenyl benzene preparation of acid

By following method Synthetic 2-hydroxyl-5-Phenylbenzoic acid.(474mg.2mmol uses Green ﹠amp with the 4-phenylphenol of tetrahydropyrans (THF) protection; The described standard method of Wats pp.31-34 is by 4-phenylphenol preparation) anhydrous THF (5ml) solution be cooled to-78 ℃.Handle with n-Butyl Lithium (hexane solution of 2ml 1.6M).Stirred reaction mixture, and be warmed to room temperature, form brown suspension therebetween.After 2 hours, reaction mixture is cooled to-78 ℃, with excessive anhydrous CO ₂Handle several minutes, stirred reaction mixture also is warmed to room temperature.After 2 hours, reaction mixture is distributed between the ether and the 1NNaOH aqueous solution.With ice water layer is cooled to 0 ℃, uses the 1NHCl acidified aqueous solution to pH2.Use methylene dichloride dispensing water solution then.Use the dried over mgso organic layer.Vacuum concentration obtains the crude product (258mg, 2-hydroxyl-5-Phenylbenzoic acid) of pale solid.The NMP spectrum of crude product is consistent with the structure of expection.

1H NMP(DMSO-d6) & 8.04(d，J＝3Hz，1H)，7.76(dd，J＝9，3Hz，1H)，7.62(d，J＝7Hz，2H)，7.44(t，J＝7Hz，2H)，7.32(t，J＝7Hz，1H)，7.00(d，J＝9Hz，1H)。

The Mass Calculation value: 466.5, the quality measured value: 467.2, LC uses 25-45% acetonitrile gradient wash-out 13 minutes.The LC retention time is 3.5 minutes.Product need not to be further purified then and can use.III. the solution of formula I compound is combined to

A. the preparation of the compound 10 of Table I and III

In in salt/ice bath, being cooled to 20g (L)-phenylalanine of-5 ℃, add the 74ml vitriol oil.Make the temperature of reaction mixture reach balance, in 5 minutes, be added dropwise to the 9.4ml concentrated hydrochloric acid then.Reaction mixture was stirred 30 minutes, add 700ml trash ice/water then.Regulate pH to 8-9 with dense ammonium hydroxide.Make solution crystallization at room temperature.Cooled off liquid at 4 ℃ then.Collect light yellow crystalline product with hardened filter paper, with cold water washing and dry.Make product recrystallization in hot water, evaporated filtrate and recrystallization (2x), overall yield is 55%.

MP:218-222 ℃ of (crude product) .TLC:R _f(F) 0.18.NMR:(D ₂O/NaOD), r2.99 (dd, J=13.4,7.2Hz, 1H), 3.10 (dd, J=13.4,6.0Hz, 1H), 3.57 (dd, J=7.2,6.0Hz, 1H), 7.48 (d, J=8.6Hz, 2H), 8.21 (d, J=8.7Hz, 2H).

The amino acid of tertbutyloxycarbonyl (BOC) protection can prepare with ordinary method.Referring to Greene ﹠amp; Wats Protectine Groups in Organic synthe-sis, 2nd E2., John Wiley ﹠amp; Sons, Inc.:New York, pp.327-328 (1991).

To the p-nitrophenyl L-Ala of-20 ℃ BOC protection (3.00g, add in anhydrous methylene chloride 9.67mmol) (5ml) solution N-methylmorpholine (NMM, 1.07ml, 9.67mmol), then add isobutyl chlorocarbonate (1.26ml, 9.67mmol).Reaction mixture was stirred 15 minutes down at-20 ℃.In mixture, add then group's methyl-L-proline hydrochlorate (L-Pro-OMe, 1.60g, 9.67mmol), then add again NMM (1.07ml, 9.67mmol).After-20 ℃ stirring was also at room temperature stirred one hour in one hour down, the vacuum concentration reaction mixture diluted with ethyl acetate then.Ethyl acetate solution 10% aqueous citric acid solution, water and salt water washing are with dried over mgso and vacuum concentration.Obtain 2.4g product () 60% productive rate behind the column chromatography purifying).

Use normal condition to remove the BOC group with the dioxan solution-treated of HCl.Referring to, Greene ﹠amp; Wuts, the same, pp328-329.To 1-hydroxyl-2-naphthoic acid (160mg, methylene dichloride 0.84mmol) and N, dinethylformamide (1: 1, add in the 3ml solution I-hydroxybenzotriazole (159mg, 1.17mmol).Solution is cooled to 0 ℃, and adding 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI, 161mg, 0.84mmol).With solution 0 ℃ stir 45 minutes after, the dipeptides that adding prepares above (300mg, 0.84mmol) solution, then add NMM (93 μ l, 0.84mmol).Reaction mixture (was stirred 1 hour, stirring at room 45 minutes at 0 ℃.Then with the reaction mixture vacuum concentration, with ethyl acetate and water dilution.Separate organic layer, water is used salt water washing (4x) then, dry and vacuum concentration.Obtain coupling product (300mg, 73%) behind the chromatogram purification.

(410mg adds glacial acetic acid (10) and 10% palladium/gac (100mg) in ethyl acetate 0.83mmol) (20ml) solution to coupling product.With solution hydrogenation 5 hours, use the diatomite filtration reaction mixture then, vacuum concentrated solution obtains 375mg (98%) product, need not to be further purified then and can use.

Product (the 200mg of hydrogenation upward, 0.45mmol) anhydrous methanol (10ml) solution in add formamidinesulfinic acid (118mg, 0.95mmol is by according to people such as Maryanoff (1986), the method of J.Org.Chem.1082 general introduction, the formamidine sulfinic acid that oxidation is bought from market and prepare).Reaction mixture was stirred 3 days, the vacuum concentration reaction mixture, product (is used methyl alcohol: the methylene dichloride wash-out) obtain the required product of 17mg, i.e. the compound 10 of Table III by chromatographic separation.IV. the alternately solution of formula I compound is combined to

A.1-thionyl chloride (15ml) solution of benzyloxy-2-naphthoic acid (2.247g) refluxed 4 hours.The reagent that vacuum-evaporation is excessive.Resistates dilutes with dry DMF (9ml) and is concentrated into about 2ml.In this solution, add 4-Dimethylamino pyridine (1.02g).Mixture at room temperature stirred spend the night.Pyridine magnesium complex with gained is added to the nitro-L-arginine (1.77g) that is stirring then, in DMF (60ml) solution of tetramethyl guanidine (1ml) and lithium chloride (4.56g).Mixture at room temperature stirred 48 hours.The most of DMF of vacuum-evaporation distributes resistates between 1.5 equivalents (N) the HCl aqueous solution and ethyl acetate.The organic layer that merges is with the saturated sodium-chloride water solution washing and use dried over mgso.Vacuum concentration obtains crude product (3.5g, 1-benzyloxy-2-naphthoyl-NG-nitro-L-arginine).TLC: the Rf of product is 0.22 (with silica-gel plate and with the chloroformic solution wash-out of 10% methyl alcohol/acetate).Product need not to be further purified then and can use.

Dry DMF (5mg) solution of 1-benzyloxy-2-naphthoyl-NG-nitro-L-arginine (150mg) is cooled to-25 ℃, and handles with isobutyl chlorocarbonate (0.053mg).Stirred reaction mixture also is warmed to room temperature.After 20 minutes.Add solid L-proline(Pro) acid amides (with its HCl salt form, 65.9mg).Mixture was stirred 16 hours, dilute with ethyl acetate then.Ethyl acetate solution washs with 5% aqueous citric acid solution, saturated sodium bicarbonate aqueous solution and saturated sodium-chloride water solution, and uses dried over mgso.Vacuum concentrated solution, with ethanol (40ml contains 10% methyl alcohol and 10% acetate) dilution resistates, with 10% palladium/charcoal (10mg) at 52 pounds of 1 square inch of psi) time hydrogenation finishes until reaction and (carries out TLC with silica gel, use chloroform: 9: 1 wash-outs of methyl alcohol).Shown in quick yield point disappearance.The reaction mixture diatomite filtration is with ethanol and methanol wash.Vacuum concentrated mixture obtains 1-hydroxyl-2-naphthoyl-L-arginyl-L-proline(Pro) acid amides (41mg, EFI mass spectrum: M with the high pressure liquid chromatography purifying ^{+ 1}=441), separate with its trifluoroacetate.

B. the preparation of the compound 1 of Table III

According to the method for above-mentioned part A, replace 1-benzyloxy-2-naphthoic acid with 2-benzyloxy-1-naphthoic acid, replace L-proline(Pro) acid amides with the sarkosine acid amides, make the compound 1 of Table III.

C. the preparation of other formula I compound

According to the method for above-mentioned part B, replace the sarkosine acid amides with following compounds:

L-proline(Pro)-N, the N-dimethylformamide;

The L-proline methyl ester; And

The trans-4-hydroxy-l-proline methyl esters; Make following compounds:

The compound 5 of Table I and II;

The compound 8 of Table I and II; And

The compound 4 of Table III.V. physical data

Table I

Compound	The Mass Calculation value	Quality measured value (M +1) 1	The LC gradient 2	LC retention time (branch)
					1	414.24	415.1	D	5.8
2	404.26	405.5	B	11.5
					3	440.26	441.1	A	10.3
4	471.26	472.2	D	6.2
					5	468.29	469.1	D	6.8
6 3	551.34	552.3	D	7.5
					7	551.34	552.1	D	7.3
8	455.26	456.0	D	7.0
					9	442.26	443.0	D	5.5
10	-	-	-	-
					11	456.26	456.7	C	24.4
12	440.26	441.2	C	25.4
					13	587.34	588.0	C	26.9
14	440.26	440.5	A	9.0
					15	440.26	440.2 4	B	8.4
16	391.5	391.8 4	D	5.6
					17	456.49	456.7 4	B	9.0
18	393.22	391.8 4	B	5.5
					19	390.4	391.1	A	4.7
20	406.4	407.2	C	20.7
					21	466.5	467.2	E	3.5

1. measure all quality with Finnigan EFI mass spectrograph, except as otherwise noted.

2. use the binary gradient system of forming by acetonitrile and water in all cases.All eluents contain 0.1% trifluoroacetic acid.The A:20-45% acetonitrile lasts 13 minutes.The BV:10-35% acetonitrile lasts 13 minutes.The C:5-95% acetonitrile lasts 45 minutes.The D:2-95% acetonitrile lasts 10 minutes.The E:25-45% acetonitrile lasts 13 minutes.

3. substances is the mixture of diastereomer.

4. with Biolon mass spectrograph quality measurement, its value representation M ⁺VI. external tryptase inhibition test

To be dissolved in methyl-sulphoxide (DMSO) to the compound (approximately 1mg) of tryptase test, and with containing 50mMn Tris-HCL (pH8.2), the damping fluid of 100mMNaCl and 0.05%Tween-20 is diluted to 1: 10.Prepare 7 parts of 3 times of other diluents with the same damping fluid of adding 10%DMSO by initial diluent.Per 1 part aliquots containig (50 μ l) of 8 parts of diluents is forwarded to successively in each hole of micro-titration plate at the bottom of the 96 hole U.Then with tryptase (25 μ l; 0.5nm ultimate density) be added in each hole, and with sample mixed, at room temperature chamber light (promptly in experimentation indoor sting light) or under " illumination " or " dark " condition of control, cultivated 1 hour." illumination condition " of this paper is meant the illuminance of 400～1500 ft-cs that luminescent lamp sends, and this paper " dark " is meant with aluminium foil and covers sample receiver.Add synthetic three peptide substrates then, tosyl group-Glyprolys-p-Nitroaniline (25 μ l; 0.5mM ultimate density) beginning enzyme reaction is at once transferred to UU/MAX kinetics microplate registering instrument (Molecular devices) with the micro-titration plate, then hydrolysis chromogen substrate 5 minutes when spectrophotometric is 405nm.Enzyme test obtains linear curve of progress usually under these conditions.Calculate the initial velocity value with degree in the dynamic analysis that is called " Batchki " (or from BiokinLed., Madison WI buys) by curve of progress, this initial velocity value is used for determining the apparent inhibition constant of fixed every inhibition.Said procedure is designed to finish the recurrence and the fitting of a curve of nonlinear data.

Table II is to having gone out the inhibition constant that several The compounds of this invention is surveyed (K ' i, μ M), the R in these compounds ¹Be hydrogen; R ²Be hydrogen; R ³For-(CH ₂) ₃-NH-(C=NH)-NH ₂R ⁴And R ⁵Form 5 yuan of heterocycles with nitrogen and carbon that they connected; And R ⁶Be hydrogen.According to the present invention, as the K ' i of compound during less than 1000 μ M, compound is called as " active " or effective tryptase inhibitors.Different with Ki, K ' i is not that of enzyme inhibitors title complex is true irrelevant often in noncompetitive inhibitor, K _i' wait K _iFor competitiveness and noncompetitive inhibitor, K _i' with K _iBe directly proportional.

The test(ing) medium solution exposure of test compound can be reduced K _i'.Usually, test conditions is light activated, and room light changes the repeatability that can influence test according to trial-production.Because this potential source of error, some tests are then carried out under dark condition.The K that in Table II and III, is arrived _iThere is not under the optical condition of chamber, measuring of bracket.The K that bracket is arranged _i' be to measure under " dark " condition that defines in the above.Have asterisk ( ^*) K _i' be that (promptly under the illuminance of 400-450 ft-c) measured under " illumination " condition that defines in the above.

Table II

Table III has been listed the inhibition constant (K that several The compounds of this invention is surveyed _i', μ M).According to the present invention, as the K of compound _i' during less than 1000 μ M, compound is called as " active " or effective tryptase inhibitors.

Table III

VII. the compound of Table I and II suppresses other characteristic of tryptase

The A.pH dependency

The inhibition of 3 pairs of tryptases of test compound in the buffer system of following four kinds of different pH: 120mM NaCl, 2.7mM KCl, 0.13mMNaH ₂PO ₄, 0.896mM Na ₂HPO ₄Used final buffer system comprises 0.05%Tween-20.Table IV is represented the K of compound 3 under different pH _i' (μ M).

Table IV

pH	6.5	7.0	7.5	8.0
					K i′(μM)	14	8.0	5.4	6.1

The best test during B.pH7.5

The inventor has found that 3 pairs of light of compound are more insensitive in following buffer system: 120mM NaCl, 2.7mM KCl, 0.13mM NaH ₂PO ₄, 0.896mM Na ₂HPO ₄With 0.05%Tween-20pH be 7.5.Therefore, when finishing above-mentioned testing sequence, the dilution of compound 3 and the adding of tryptase all can be carried out under chamber light room temperature (22-26 ℃).Before adding substrate, cover test with aluminium foil and cultivated 1 hour, this cultivation also can be carried out under the light of chamber.The step of dilution and adding tryptase must be finished in 5 minutes.In addition, compound 3 must be dissolved among the DMSO.VIII. in vivo test

In these researchs, use the allergy sheep model of asthma.Disclose before this these methods (referring to people such as Abraham, (1983) Am.Rev.Respir.Dis.128:83-844; People such as Allegra (1983) J.Appl.Physiol.59:1416-1422; Every sheep of people such as Soler (1989) J.Appl.Physiol.67:406-413. all as itself in the same old way.The body weight of these animals is the 20-50 kilogram.

In these researchs, the compound 3 of 9mg Table I and II is dissolved in the 3mL buffer saline, before antigenic stimulation 0.5 hour, 4 hours afterwards and 24 hours afterwards, with total solution with the aerosol form administration.(total dose=27: n=6), compound 3 shows to alleviate warming up of replying in early days and can reduce the later stage and replys as shown in Figure 1.In contrast (only using carrier) test, antigenic stimulation makes the peak value in early stage and later stage surpass baseline, and specific lung hinders (the SRL) (mean value ± SE) that increases to 374 ± 104% and 212 ± 21% respectively.In contrast, when handling sheep with Table I and II compound 3, the SRL early stage and later stage increases to 280 ± 39% and 72 ± 9% (P＜0.05, and to compare in the same old way, the Wilcoxon signal is arranged and tested).Reply the peaked mean value of peak value in early days for stimulate the back to produce at once.Thereby the maximum response value that every animal is obtained on average calculates the later stage replys peak value.This method is reliably, and it has been got rid of owing to simple average, and the later stage replys and may reduce.In contrast and drug test after the antigenic stimulation 24 hours, the strong responsiveness of sheep generation air flue.(the strong responsiveness of air flue can be expressed as PC400, that is, make SRL increase by 400% long crust can concentration.Therefore, PC400 reduce show that air flue becomes strong responsiveness).The compound 3 of Table I and II has stoped 24 hours strong responsiveness.Referring to Fig. 2.Baseline PC400 is 22. ± 3.7 breathing units in controlled trial, reduces to 9.1 ± 1.3 and breathe units (compare with baseline P＜0.05) after antigenic stimulation.In contrast, in drug test, do not change (16.0 ± 3.8 17.63.5 that breathe behind unit and the antigen stimulation breathe unit relatively) with respect to baseline PC400.Therefore, the air flue function of handling the sheep that makes antigen stimulation with Table I and II compound 3 produces statistically evident improvement.

Disclosed in this application all papers and reference comprise that patent is hereby incorporated by.

Certainly describe above is to be used for explanation rather than restriction.Many embodiments are apparent for having read above-described those skilled in the art.Therefore, scope of the present invention should not determined according to foregoing description, and should determine according to appended claim and with the corresponding gamut of these claims.

Claims (16)

1. following formula: compound or its pharmaceutically acceptable salt,

Wherein:

Ar is the aryl of hydroxyl replacement or the heteroaryl that hydroxyl replaces, and wherein hydroxyl is positioned at the ortho position of amide side chains, if Ar is the aryl that hydroxyl replaces, the aromatic ring that then has amide side chains is not replaced and do not have low alkyl group on the ortho position of hydroxyl by halogen;

R ¹Be hydrogen, low alkyl group, arylalkyl or heteroarylalkyl;

R ²Be hydrogen or low alkyl group;

R ³Be selected from:

Wherein m is the integer of 3-6, and p is the integer of 0-2, and q is the integer of 0-2; R is the integer of 0-5; S is the integer of 0-2; U is 1 or 2; A is-CH=CH-or-C ≡ C-; X is-NH-;

R ⁴Be low alkyl group, the arylalkyl of replacement or the heteroarylalkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroarylalkyl of replacement; Or R ⁴And R ⁵4 yuan, 5 yuan or 6 yuan of heterocycles forming replacement with nitrogen that they connected and carbon; R ⁶Be hydrogen; Or R ⁴And R ⁶With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁵Be hydrogen;

R ⁷For-OR ⁸Or-NR ⁸R ⁹, R wherein ⁸And R ⁹Independently be selected from hydrogen, low alkyl group, aryl, arylalkyl or heteroarylalkyl, or R ⁸And R ⁹Form 5 yuan or 6 yuan of heterocycles of replacement with the nitrogen that they connected.

2. according to the compound of claim 1, wherein

Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridyl or 2-hydroxyl-3-quinoxalinyl;

R ¹Be hydrogen;

R ²Be hydrogen;

R ³Be selected from:

Wherein m is the integer of 3-6, and p is the integer of 0-2, and q is the integer of 0-2, A is-CH=CH-or-C=C-; X is-NH-;

R ⁴Be low alkyl group, R ⁵And R ⁶Be hydrogen; Or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen;

R ⁷For-OH ,-OCH ₃,-NH ₂, 3 '-amino carboxyl-1 '-piperidyl or-N (CH ₃) ₂

3. according to the compound of claim 2, wherein Ar is 1-hydroxyl-2-naphthyl, R ¹Be hydrogen, R ²Be hydrogen, R ³For-(CH ₂) ₃NH (CNH) NH ₂, R ⁴And R ⁵With 5 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen and R ⁷For-NH ₂

4. according to the compound of claim 2, wherein Ar is 2-hydroxyl-1-naphthyl, and R ' is a hydrogen, R ²Be hydrogen, R ³For-(CH ₂) ₃NH (CNH) NH ₂, R ⁴And R ⁵With 5 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen, R ⁷For-NH ₂

5. treat the aerosol combination of immunoregulatory inflammation, comprise the compound that is in the claim 1 in pharmaceutically acceptable aerosolization carrier soln or the dry powder.

6. the composition of claim 5, wherein:

Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridyl or 2-hydroxyl-3-quinoxalinyl;

R ¹Be hydrogen;

R ²Be hydrogen;

R ³Be selected from:

Wherein m is the integer of 3-6, and p is the integer of 0-2, and q is the integer of 0-2; A is-CH=CH-or-C ≡ C-; X is-NH-;

R ⁴Be low alkyl group, R ⁵And R ⁶Be hydrogen; Or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen;

R ⁷For-OH ,-OCH ₃,-NH ₂, 3 '-amino carboxyl-1 '-piperidyl or-N (CH ₃) ₂

7. the composition of claim 6, wherein Ar is 1-hydroxyl-2-naphthyl, R ¹Be hydrogen, R ²Be hydrogen, R ³For-(CH ₂) ₃NH (CNH) NH ₂, R ⁴And R ⁵With 5 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen and R ⁷For-NH ₂

8. the composition of claim 6, wherein Ar is 2-hydroxyl-1-naphthyl, R ¹Be hydrogen, R ²Be hydrogen, R ³For-(CH ₂) ₃NH (CNH) NH ₂, R ⁴And R ⁵With 5 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen and R ⁷For-NH ₂

9. the composition of claim 5, the inflammation of wherein said respiratory tract is an asthma.

10. the composition of claim 5, the inflammation of wherein said respiratory tract is rhinallergosis.

11. the composition of claim 5, wherein the described compound of claim 1 is present in the described carrier soln with the concentration of 0.1-30mg/ml.

12. pharmaceutical composition comprises and pharmaceutically acceptable carrier-bound claim 1 compound.

13. the composition of claim 12, wherein pharmaceutically acceptable carrier comprises nontoxic pharmaceutically acceptable topical carrier.

14. the composition of claim 12, wherein

Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridyl or 2-hydroxyl-3-quinoxalinyl;

R ¹Be hydrogen;

R ²Be hydrogen;

R ³Be selected from:

Wherein m is the integer of 3-6, and p is the integer of 0-2, and q is the integer of 0-2; A is-CH=CH-or-C ≡ C-; X is-NH-;

R ⁴Be low alkyl group, R ⁵And R ⁶Be hydrogen; Or R ⁴And R ⁵With 4 yuan, 5 yuan or 6 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen;

R ⁷For-OH ,-OCH ₃,-NH ₂, 3 '-amino carboxyl-1 '-piperidyl or-N (CH ₃) ₂

15. the composition of claim 14, wherein Ar is 1-hydroxyl-2-naphthyl, R ¹Be hydrogen, R ²Be hydrogen, R ³For-(CH ₂) ₃NH (CNH) NH ₂, R ⁴And R ⁵With 5 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen and R ⁷For-NH ₂

16. the composition of claim 14, wherein Ar is 2-hydroxyl-1-naphthyl, R ¹Be hydrogen, R ²Be hydrogen, R ³For-(CH ₂) ₃NH (CNH) NH ₂, R ⁴And R ⁵With 5 yuan of heterocycles that nitrogen that they connected and carbon form replacement, R ⁶Be hydrogen and R ⁷For-NH ₂

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