CN1234229A - Apparatus for treatment of immunomediated inflammatory disorders - Google Patents

Abstract

Compositions and methods for the prevention and treatment of immunomediated inflammatory disorders, especially for those disorders associated with the respiratory tract, are provided. 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, as well as aerosol devices for administering these compositions intranasally.

Description

The device of treatment immunomediated inflammatory disorders

The application is dividing an application of No. 94191441.0 patent applications, and the applying date of original application is on March 11st, 1994, and denomination of invention is " treating the compositions and the method for immunoregulatory inflammation ".

The present invention relates generally to prevent and treat the compositions 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 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 is dwindled and the strong responsiveness of air flue.

But have now found that inhibitory enzyme particularly the low molecular weight compound of protease can be widely used in the treatment various pathologic conditions.Many these compounds are called as peptide mimics, because as their name referring, these chemical compounds have simulated the various inhibitor peptides of only being made up of condensation aminoacid.The mimic protease inhibitor of these low molecular peptides is compared the special benefits that has with native peptides and is 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 Angiotensin-Converting (ACE) is determining angiotensin to be split into angiotensin.Suppressing ACE is the hypertensive method of a kind of control.ACE can be suppressed effectively by the sulfydryl acyl derivative of proline, 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 and analog 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 probability of apoplexy.Thrombin can particularly be called arginic amino acid derivativges by many peptide mimicses and substitute 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 syndrome, 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 bronchioles 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 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 leukocyte that has an IgE receptor is mastocyte and basophilic leukocyte but also comprise that mononuclear cell, macrophage 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.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 the deutero-medium of superoxides and lipid.In addition, the threshing by mastocyte can discharge several macromole: proteoglycan, peroxidase, ARB, particularly trypsinlike enzyme and chymase (chymase).Referring to " Drug Therapy ofAsthma ", chap 62,1054-54.

This chemical release of mastocyte can explain that the early stage bronchioles 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 bronchioles smooth 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 macrophage.

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 crosslinking.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, activatory mastocyte discharges the primary and secondary inflammatory mediator when threshing.Eosinocyte and macrophage attracted to above-mentioned position to keep inflammatory reaction.The destruction of nasion epithelial tissue 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.Adult's trypsinlike enzyme is the relevant tetramer of glycosylated and heparin of the activatory subunit of a kind of heterogeneous catalysis.There is not approaching corresponding thing in a large amount of other serine stretch protein acid of the same characterization of class Trypsin acid monomers aminoacid sequence with its unit structure.Referring to as people such as Vanderslice (1990) Pron.Natl.Acad.Sci.USA87:3811-3815; 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 Vanderslice (1989) Biochemistry28:4148-4155; With people (1990) Monographs in Allergy27:51-66 such as Katunuma.

Trypsinlike enzyme is stored in the mastocyte secretory granule.After the mastocyte activation, in various biofluids, be easy to measure human tryptase.For example, after anaphylaxis, trypsinlike enzyme 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 idiosyncrasy subject with specific antigen and lung-douching fluid, can measure the existence of trypsinlike enzyme.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.Trypsinlike enzyme level in the lung-douching fluid that is obtained by the idiosyncrasy asthma patient after the bronchial mucosa allergen intensifies increases.Equally, the trypsinlike enzyme 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 edema due to disorder of QI that support is provided to the release of the protease of activation mastocyte.Referring to people such as Kalenderian (1988) (chest94:119-123.In addition, proved that now trypsinlike enzyme is a kind of fibrocellular effective mitogen, thereby shown that it is relevant with a matter pneumonopathy with pulmonary fibrosis.Referring to people such as Ruoss (1991) J.Clin.Invest.88:493-499.

Trypsinlike enzyme is relevant with various bioprocesss, and the degraded that comprises the lax neuropeptide of vasodilation and bronchus is (referring to people such as Caughey (1988) J.pharmacol.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 bronchus responsiveness.These studies show that trypsinlike enzyme may increase the contraction of asthma mesobronchus by destroying the bronchiectasis peptide.

In addition, verified trypsinlike enzyme 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) trypsinlike enzyme is also relevant with reconstruction with the inflammation of tissue.This discovery also shows trypsinlike enzyme generation effect in the destruction of joint of rheumatoid arthritis.Proved that in addition trypsinlike enzyme can divide the peptide relevant with Procalcitonin..Because this peptide and neuron inflammation-related, so trypsinlike enzyme is a key 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 trypsinlike enzymes can be split into thrombin with prothrombase, and regulated the identification (referring to people such as Katununa (1990) Monographs in Allergy27:51-66) 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 therapeutic 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 compositions and the method for these diseases simultaneously effectively.

The invention provides new tryptase inhibitors, it comprises on formula I chemical 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, aryl alkyl or heteroaryl alkyl;

R ²Be hydrogen or low alkyl group;

R ³Be selected from:

-(CH ₂) _vNH ₂

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 aryl alkyl of replacement or the heteroaryl alkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroaryl alkyl 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, aryl alkyl or heteroaryl alkyl, 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-pyridine radicals 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 chemical compound 3 of table I and II: The tryptase inhibitors of another preferred formula I is the chemical compound 15 of table I and II:

Chemical 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 allergic rhinitis 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 chemical compound described herein.These pharmaceutical compositions have various dosage forms and comprise peroral 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 powder or solution.When being used for the treatment of immunoregulatory inflammation dermatosis, The compounds of this invention can be used in combination with nontoxic pharmaceutically useful topical carrier.Chemical compound of the present invention can be used in combination with the corticosteroid of antibiotic medicine or other treating asthma medicines such as beta-adrenergic antagonistic, antiinflammatory, anticholinergic etc.

Fig. 1 for the specific lung resistance of expression sheep as antigenic stimulus after the time (hour) the curve of function.Open squares represents to contrast class value, and the value of identical animal behind the chemical 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 bronchoconstrictions of long crust after the antigenic stimulus take table I and II chemical compound 2 time.The solid black bar is corresponding to matched group, benchmark.Light color solid bars corresponding to medicine, benchmark.The shade lines are corresponding to matched group, behind the antigen.White bars is corresponding to medicine, behind the antigen.

I. definition and general parameter

Following definitions is used to illustrate and define 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, urticaria, angioedema, eczematoid dermatitis (atopic dermatitis), anaphylaxis and high proliferative skin disorders, peptic ulcer, enteritis, dermatitis etc.

" strong responsiveness " is meant later stage bronchoconstriction and the airway hyperreactivity relevant with chronic asthma.The strong responsiveness of the bronchioles tissue of asthma is considered to be caused by chronic inflammatory reaction, and inflammatory reaction stimulates and operation is gone up the airway walls in the leather sheet and impelled the pathologic thickening of lower-hierarchy.

" halogen " is meant fluorine, bromine, chlorine and iodine atom.

" hydroxyl " is meant group-OH.

" low alkyl group " is meant 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 " be meant have monocycle (as phenyl), multi-ring (as xenyl) or wherein at least one ring be that many fused rings of aromatic ring are (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, the aromatic ring that has amide side chains can not further be replaced by halogen.In addition, have the aromatic ring of amide side chains and low alkyl group can not be arranged the ortho position of hydroxyl (be amide side chains between position).

" heterocycle " is meant 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 has saturated, the unsaturated or aromatic carbocyclyl groups of at least one hetero atom such as N, O or S in ring, and 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 " HetAr " are meant that wherein at least one heterocycle is the heterocycle of aromatic ring.

" aralkyl " be meant 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 " be meant 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 " is meant biological effect and the character that keeps parent compound, and is not biologically or other unwanted salt.

" medicine or treatment go up acceptable carrier " is meant and do not influence the biological activity of active ingredient effect and to host or the nontoxic mounting medium of patient.

" stereoisomer " is meant to have same molecular amount, chemical composition and structure, but atom is arranged different chemical 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 rotation that some pure stereoisomer has is very low and can not measure with modern instrument.Chemical 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 " be meant 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 compositions of tryptase inhibitors, it can be used for alleviating immunoregulatory inflammation particularly the asthma animal stimulate inductive bronchoconstriction by allergen.

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

Tryptase inhibitors comprises formula I chemical 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, aryl alkyl or heteroaryl alkyl;

R ²Be hydrogen or low alkyl group;

R ³Advance certainly:

-(CH ₂) _vNH ₂

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 aryl alkyl of replacement or the heteroaryl alkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroaryl alkyl 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, aryl alkyl or heteroaryl alkyl, 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-pyridine radicals 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 chemical compound 3 of table I and II:

Another preferred tryptase inhibitors is the chemical compound 15 of table I and II:

Another preferred tryptase inhibitors is the chemical compound 21 of table I and II:

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

The character that depends on functional group, The compounds of this invention can form addition salts with various inorganic and organic bronsted lowry acids and bases bronsted lowries.Form mineral acid example hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid of these salt etc., organic acid such as acetic acid, propanoic acid, glycolic, acetone acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid etc.

Also can pass through with alkali 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 hydroxide and alkaline-earth alkoxides processing hydroxy-acid group generation salt.In addition, also can generate salt, organic base such as trimethylamine, diethylamine, ethanolamine, piperidines, 2-aminopropane., choline, caffeine etc. by carboxylic acid and organic base.

These salt can prepare with conventional 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 lyophilization such as the water, or to make the cation exchange of existing salt on suitable ion exchange resin be other cation.

III. external and in vivo test

The in vitro tests method of screening effective inhibitors according to the ability that suppresses trypsinlike enzyme is known in the prior art.For example referring to people such as Sturzebecher (1992) Biol.Chem.Hoppe-Seyler373:1025-1030.Usually, the hydrolysis of the inductive peptidyl chromogen substance of these experimental measurement trypsinlike enzymes.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 Models of ReversibleAirway obstruction ", The Lung:Scientific Foundations, 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 feature, comprise: to the strong responsiveness of air flue of chemical mediator and physical stimulation, the medicine (β-adrenergic, methylxanthine, corticosteroid etc.) that is used for people's asthma can make airway obstruction reverse; Activatory leukocytic infiltration produces airway inflammation; The degeneration of chronic inflammatory disease changes, for example basement membrane thickening, smooth muscle hypertrophy and epithelial damage.The kind that can be used as animal model comprises mice, Mus, Cavia porcellus, rabbit, Canis familiaris L. and sheep.All animals all have some restriction, but select animal model to depend on related problem suitably.

With Cavia porcellus and Canis familiaris L. 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 allergia model.The compositions of using the aerosolization solution that contains The compounds of this invention for before or after being exposed to specific allergen the allergia sheep proves that these compositionss can alleviate greatly or get rid of that later stage asthma is replied and strong responsiveness subsequently.

Chemical compound of the present invention also can be used for treating other immunoregulatory inflammation that trypsinlike enzyme activity wherein causes pathological state.These diseases comprise the inflammation relevant with mastocyte such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritis disease, enteritis, peptic ulcer and various dermatosis.

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 therapeutical effect of various dermatosiss such as high proliferative skin disorders, for example Semen arachidis hypogaeae device olefin(e) acid Mus 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 Internation-ales de Pharmacodynamie et de Therapie270:128-140.

IV. vivo medicine-feeding

According to the present invention, give particularly formula I chemical compound of tryptase inhibitors that the patient suffer from immunoregulatory inflammation takes treatment or medicine effective quantity.According to a specific embodiments, compositions 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), anaphylaxis and high proliferative skin disorders, peptic ulcer etc.

The compositions that contains The compounds of this invention can preventative and/or curative form administration.In treatment is used, take the compositions of curing or suppressing disease symptoms and complication effective dose 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 effective dose or dosage ".Effective dose used herein depends on the order of severity of disease and stadium, former treatment, patient's health status, 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 compositions that contains The compounds of this invention.So consumption is defined as " prevention effective dose or dosage ", and in this used, accurate consumption also depended on patient's health status, 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 disease, treatment between patient needs for a long time.

Usually, the suitable effective dose of tryptase inhibitors is 0.1 to 1000 milligram (mg)/receptor/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 forms.For example the per unit dosage form contains 5 to 1000mg preferred 10 to 100mg active ingredients.

Compositions for use can adopt various dosage forms in these treatments.They comprise solid, semisolid and liquid dosage form, as tablet, pill, powder, liquid solution or suspension, liposome, injection and infusion liquid.Preferred dosage form 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 effective dose 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 ofTherapeutics, 8th Ed. is among Pergamon Press and the Remington ＇ s Supra.Discussed in the document that medication is for example oral, intravenous, intraperitoneal or intramuscular administration and other method.Pharmaceutically suitable carrier comprises water, saline, buffer and at the Merck Index, Merck﹠amp; Co.Rahway, other chemical compound of describing among the NJ..

Usually, when The compounds of this invention is used for the treatment of asthma or allergic rhinitis, they are mixed with aerosol.Term " aerosol " comprise The compounds of this invention the suspension media that can produce gas, it can be inhaled into bronchioles 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 aerosol apparatus.Aerosol also comprises the powdered compositions that is suspended in the The compounds of this invention in air or other vector gas, combines by inhalation from inhaler.

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 buffer that allows on the physiology such as phosphate 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 chloride that adds is adjusted to biological scope with permeability, preferably in 10% isotonicity.For the preparation that produces these solution that aerosol sucks has come into question in Remington ＇ s Pharmaceutical Sciences, also can be referring to Ganderton﹠amp; Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems6:273-313; With people (1992) J.pharmacol.Toxicol.Methods27:143-159 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 pressue 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 trachea 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 trachea.

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 allergic rhinitis, the inventive system comprises the solution of The compounds of this invention is loaded in the jetmizer.

The dry powder doses of the excipient 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 inhaler administration of above-mentioned powder.

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 (fanoterol) or prenaline.

Other medicament that is used in combination with The compounds of this invention comprises that the scorching sterin of cortex (adrenocortical steroid) of anticholinergic agent such as the antiinflammatory of ipratropium bromide group is as beclomethasone, omcilon, flurisolide or dexamethasone.

Chemical compound of the present invention also can be used for treating mammiferous immunoregulatory inflammation dermatosis such as urticaria and angioedema, eczematoid dermatitis and high proliferative skin disorders such as psoriasis.Topical by the formula I chemical compound is expected mitigation symptoms.Therefore suffering from the dermopathic patient of immunoregulatory inflammation is expected to alleviate scale, erythema, speckle size, pruritus and other symptom relevant with dermatosis.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 chemical compound is 0.0001% to 10% and the carrier of nontoxic pharmaceutically useful local usefulness.These topical formulations can prepare by conventional medicine diluent 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 gel is prepared with water base or oil base.These substrate comprise that water and/or oil are as liquid Halloysitum Rubrum or vegetable oil such as Oleum Arachidis hypogaeae semen or Semen Ricini oil.Comprise sunshine, aluminium stearate, cetyl octadecanol, propylene glycol, Polyethylene Glycol, lanoline, hydrogenant lanoline, Cera Flava etc. according to the available thickening agent of the character of substrate.

Washing liquid can be prepared with aqueous or oily substrate, and it generally also comprises one or more following compositions: stabilizing agent, emulsifying agent, dispersant, suspending agent, thickening agent, coloring agent, spice etc.

Powder can be by means of preparations such as any suitable powder substrate such as Talcum, lactose, starch.Drop can be prepared with hydrated matrix or non-aqueous matrix, and it also can comprise one or more dispersants, suspending agent, solubilizer etc.

Local medicine composition of the present invention also comprises one or more antiseptic or bacteriostatic agent for example methyl hydroxybenzoate, nipasol, chlorocresol, benzalkonium chloride etc.Local medicine composition also can contain other live bosom composition such as antimicrobial particularly antibiotics, anesthetis, analgesic and antipruritic.

Chemical 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 ulcer, 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 therapeutic agent, for example antibiotic medicine and/or analgesic such as aspirin, indometacin, BUTE, ibuprofen, naproxen, 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 forms administrations.For example, be suitable for oral unit dosage forms and comprise powder, tablet, pill, capsule and dragee.

Pharmaceutical composition of the present invention can intravenous administration.Therefore, the invention provides the intravenous administration compositions, it comprises and is dissolved in or is suspended in chemical 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% saline etc.The known sterilizing methods of the available sometimes routine of these compositionss is sterilized or is carried out aseptic filtration.The aqueous solution of gained is comprised use, for example carry out lyophilizing, before using, freeze dried preparation is mixed with aseptic aqueous solution, for reaching proximate physiological condition, these compositionss can contain pharmaceutically useful auxiliary substance such as PH adjusting and buffer agent, strong shape regulator, gentle dose etc., for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleyl alcohol salt etc.

For solid composite, can use conventional non-toxic solid carrier, it comprises mannitol, lactose, starch, magnesium stearate, saccharin sodium, Talcum, cellulose, glucose, sucrose, magnesium carbonate of pharmaceutical grade etc.For oral administration, pharmaceutically acceptable non-toxic composite can prepare by adding any excipient 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.

The experiment I. general introduction

The initiation 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.The formula I chemical compound can be combined to the technology preparation with solid or solution by method well known in the art.The initiation material of preparation 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 Peptide 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.l, (eds.Gross, E. ， ﹠amp; .J.Meienhofer).Among the Academic Press Orlando (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 technical staff 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 purification of chemical compound as herein described and intermediate.For example filter, extraction, crystallization, post color are 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., PierceChemical Company, P.114.Preparation contains 95% alcoholic solution I of 0.01% alpha-Naphthol and 5% urea.The 2g bromine is dissolved in preparation solution II in the 100ml8% sodium hydrate aqueous solution.In the solution I, add 5 sodium hydroxide.With sample point to be analyzed on thin layer chromatography (TLC) plate.Launch the TLC plate with the solution I then.The TLC plate at air drying, is launched with the solution II then.Red point shows has amino imino methane group to exist.II. the solid phase synthesis of formula I chemical compound

A. show the preparation of the chemical compound 3 of 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 be suspended in 30ml1: the N of 1 (v/v), N dimethyl formamide (DMF): contain in the solution of toluene of 30% (volume) piperidines.Reactant 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 dichloromethane (6x) washing resin granule successively.

With Fmoc-L-proline (Milligen; 6.06mmol), benzotriazole-1-base-oxygen-three (dimethylamino) hexafluorophosphate (BOP, Novabiochem; 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 granule 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-diisopropyl carbodiimides (DIPCDI; Aldrich; 0.949ml, 6.06mmol) and HOBT (0.819g, DMF 6.06ml) (45mL) solution stirred (mechanical agitation bar) 7 hours with reactant mixture.Filter, washing and handle (as stated above) with piperidines after, use DMF (6x), dichloromethane (6x) and methanol (6x) washing resin successively.Resin is suspended in the methanol, and stirs (mechanical agitation 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 phenyl ethers anisole: water (90: 5: 5,30ml) solution.Resin becomes pink at once, and color change melt into is peony 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 response time 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).Grease is dissolved in the crude product (471mg) that the fine aqueous solution of 50% second (100ml) and lyophilization obtain pale solid.

HPLC analyze (Polymer Labs100A 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 unification compound (retention time is 10.35 minutes, and UV absorbs＞98%) substantially, by HPLC C ₁₈Silicagel column (Vydac; 22 * 250mm, 15-20 μ m, 300A,, flow velocity 10mL/min with 20 to 35% acetonitrile linear gradient elutions 30 minutes) carry out purification.Need multiple injection 30-35mg, merge same fraction (retention time is about 44-48 minute) and lyophilization and obtain loose white solid (351mg), analyzing through HPLC is the unification 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.

¹H 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 chemical compound

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

2-hydroxy-4-methyl benzoic 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 benzodiazine) 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 chemical compound 2 of table I and II;

The chemical compound 9 of table I and II;

The chemical compound 14 of table I and II;

The chemical compound 15 of table I and II;

The chemical compound 16 of table I and II;

The chemical compound 17 of table I and II;

The chemical compound 18 of table I and II;

The chemical compound 19 of table I and II;

The chemical compound 20 of table I and II; And

The chemical compound 21 of table I and II;

C. the preparation of other formula I chemical compound

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

D. the preparation of other formula I chemical compound

The preparation of the chemical compound 13 of table I and II is the method that is used for coupling Fmoc-L-proline 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, finish chemical compound 13 synthetic remainders with the described method of above-mentioned part A.

Equally, the chemical 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, finish synthetic remainder 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 Pentamethylene oxide. (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-BuLi (hexane solution of 2ml1.6M).Stirred reaction mixture, and be warmed to room temperature, form the brown suspension therebetween.After 2 hours, reactant mixture is cooled to-78 ℃, with excessive anhydrous CO ₂Handle a few minutes, stirred reaction mixture also is warmed to room temperature.After 2 hours, reactant mixture is distributed between ether and 1NNaOH aqueous solution.With ice water layer is cooled to 0 ℃, uses the 1NHCl acidified aqueous solution to pH2.Use dichloromethane 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 eluting 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 chemical compound is combined to

A. show the preparation of the chemical compound 10 of I and III

In in salt/ice bath, being cooled to 20g (L)-phenylalanine of-5 ℃, add the 74ml concentrated sulphuric acid.Make the temperature of reactant mixture reach balance, in 5 minutes, be added dropwise to the 9.4ml concentrated hydrochloric acid then.Reactant 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), gross production rate is 55%.

MP:218-222 ℃ (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 aminoacid of tertbutyloxycarbonyl (BOC) protection can prepare with conventional method.Referring to Greene﹠amp; Wats Protectine Groups in Organic synthesis, 2nd E2., JohnWiley﹠amp; Sons, Inc.:New York, pp.327-328 (1991).

To the p-nitrophenyl alanine 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).Reactant mixture was stirred 15 minutes down at-20 ℃.Methyl-(L-Pro-OMe, 1.60g 9.67mmol), then add NMM (1.07ml, 9.67 mmol) to the L-proline hydrochlorate again to add group then in mixture.After-20 ℃ stirring was also at room temperature stirred one hour in one hour down, the vacuum concentration reactant 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 purification.

Use normal condition to remove the BOC group with the dioxanes solution-treated of HCl.Referring to, Greene﹠amp; Wuts, the same, pp328-329.To 1-hydroxyl-2-naphthoic acid (160mg, dichloromethane 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).Reactant mixture (was stirred 1 hour, stirring at room 45 minutes at 0 ℃.Then with the reactant 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/active carbon (100mg) in ethyl acetate 0.83mmol) (20ml) solution to coupling product.With solution hydrogenation 5 hours, use the diatomite filtration reactant 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) absolute 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).Reactant mixture was stirred 3 days, the vacuum concentration reactant mixture, product (is used methanol: the dichloromethane eluting) obtain the required product of 17mg, promptly show the chemical compound 10 of III by chromatographic isolation.IV. the alternately solution of formula I chemical 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.Residue dilutes with dry DMF (9ml) and is concentrated into about 2ml.In this solution, add 4-dimethylamino naphthyridine (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 residue between 1.5 equivalents (N) 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 eluting of 10% methanol/acetic acid).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 amide (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% methanol and 10% acetic acid) dilution residue, 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 eluting of methanol).Shown in quick pour point disappearance.The reactant mixture diatomite filtration is with ethanol and methanol wash.Vacuum concentrated mixture obtains 1-hydroxyl-2-naphthoyl-L-arginyl--L-proline amide (41mg, EFI mass spectrum: M with the high pressure liquid chromatography purification ^{+ 1}=441), separate with its trifluoroacetate.

B. show the preparation of the chemical compound 1 of 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 amide with the sarcosine amide, make the chemical compound 1 of table III.

C. the preparation of other formula I chemical compound

According to the method for above-mentioned part B, replace the sarcosine amide with following compounds:

L-proline-N, the N-dimethylformamide;

The L-proline methyl ester; And

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

The chemical compound 5 of table I and II;

The chemical compound 8 of table I and II; And

The chemical compound 4 of table III.V. physical data

The table I

Chemical 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	????440.24	?????B	?????8.4
???16	?????391.5	????391.84	?????D	?????5.6
					???17	?????456.49	????456.74	?????B	?????9.0
???18	?????393.22	????391.84	?????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 eluant 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 trypsinlike enzyme inhibition test

To be dissolved in dimethyl sulfoxine (DMSO) to the chemical compound (approximately 1mg) of trypsinlike enzyme test, and with containing 50mMn Tris-HCL (pH8.2), the buffer of 100mM NaCl and 0.05%Tween-20 is diluted to 1: 10.Prepare 7 parts of 3 times of other diluents with the same buffer of adding 10%DMSO by initial diluent.Per 1 part aliquot (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 trypsinlike enzyme (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 foot candles that fluorescent lamp sends, and this paper " dark " is meant with aluminium foil and covers sample container.Add synthetic three peptide substrates then, tosyl-Glyprolys-paranitroanilinum (25 μ l; 0.5mM ultimate density) beginning enzyme reaction is at once transferred to UU/MAX kinetics microplate monitor (Moleculardevices) 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 Biokin Led., 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 curve fitting of nonlinear data.

The 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 chemical 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 chemical compound during less than 1000 μ M, chemical compound is called as " active " or effective tryptase inhibitors.Different with Ki, K ' i is not the true irrelevant normal dried noncompetitive inhibitor of of enzyme inhibitor coordination compound, 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, experimental condition 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 foot candle) measured under " illumination " condition that defines in the above.

The table II

The 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 chemical compound _i' during less than 1000 μ M, chemical compound is called as " active " or effective tryptase inhibitors.

The table III

VII. the chemical compound of table I and II suppresses other characteristic of trypsinlike enzyme

The A.pH dependency

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

The 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 chemical 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 test procedure, the dilution of chemical compound 3 and the adding of trypsinlike enzyme all can be carried out under chamber light room temperature (22-26 ℃).Before adding substrate, cover bread board 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 trypsinlike enzyme must be finished in 5 minutes.In addition, chemical compound 3 must be dissolved among the DMSO.VIII. in vivo test

In these researchs, use the allergia 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; People such as Soler (1989) J.Appl.Physiol.67:406-413.Every sheep all as itself in the same old way.The body weight of these animals is the 20-50 kilogram.

In these researchs, the chemical compound 3 of 9mg being shown I and II is dissolved in the 3mL buffer saline, before antigenic stimulus 0.5 hour, 4 hours afterwards and 24 hours afterwards, with total solution with the aerosol form administration.(accumulated dose=27:n=6), chemical compound 3 shows and alleviates warming up and reducing the later stage of replying in early days and reply as shown in Figure 1.In contrast (only using carrier) test, antigenic stimulus makes the peak value in early stage and later stage surpass baseline, and specific lung hinders (the SRL) (meansigma methods ± SE) that increases to 374 ± 104% and 212 ± 21% respectively.In contrast, when handling sheep with table I and II chemical 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 meansigma methods 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 stimulus 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 chemical compound 3 of table I and II has stoped 24 hours strong responsivenesss.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 stimulus.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 chemical compound 3 produces statistically evident improvement.

Disclosed in this application all papers and list of references 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 claim.

Claims (14)

1. aerosol device comprises:

Chemical compound shown in the following formula in pharmaceutically acceptable carrier solution or the dry powder or its pharmaceutically acceptable salt and described solution or dry powder changed into the device that is fit to the aerosol form that sucks

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 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, aryl alkyl or heteroaryl alkyl;

R ²Be hydrogen or low alkyl group;

R ³Be selected from:

-(CH ₂) _vNH ₂

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 aryl alkyl of replacement or the heteroaryl alkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroaryl alkyl 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, aryl alkyl or heteroaryl alkyl, or R ⁸And R ⁹Form 5 yuan or 6 yuan of heterocycles of replacement with the nitrogen that they connected.

2. the device of claim 1, wherein

Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridine radicals 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, and 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

3. the device 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. the device of claim 2, 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

5. the device of claim 1, wherein said chemical compound is present in the described carrier solution with the concentration of 0.1-30mg/ml.

6. the device of claim 1 also comprises beta-adrenergic agonist chemical compound.

7. the device of claim 6, wherein said beta-adrenergic agonist chemical compound are selected from salbutamol, terbutaline, good fortune make a mistake alcohol (formoterol), fenoterol (fenoterol) and prenaline.

8. the device of claim 1 also comprises the corticosteroid of antiinflammatory.

9. the device of claim 8, the corticosteroid of wherein said antiinflammatory is selected from beclomethasone, omcilon, flurisolide and dexamethasone.

10. the device of claim 1 also comprises ipratropium bromide.

11. a device for the treatment of allergic rhinitis comprises:

Chemical compound shown in the following formula in the pharmaceutically acceptable carrier solution or its pharmaceutically acceptable salt and described solution is changed into the device of the aerosol form that is fit to intranasal administration 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 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, aryl alkyl or heteroaryl alkyl;

R ²Be hydrogen or low alkyl group;

R ³Be selected from:

-(CH ₂) _vNH ₂

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 aryl alkyl of replacement or the heteroaryl alkyl of replacement, R ⁵And R ⁶Independently be selected from: the aralkyl of hydrogen, low alkyl group, replacement and the heteroaryl alkyl 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, aryl alkyl or heteroaryl alkyl, or R ⁸And R ⁹Form 5 yuan or 6 yuan of heterocycles of replacement with the nitrogen that they connected.

12. the device of claim 11, wherein

Ar is 1-hydroxyl-2-naphthyl, 2-hydroxyl-1-naphthyl, 3-hydroxyl-2-pyridine radicals 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, and 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

13. the device of claim 12, 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

14. the device of claim 12, 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