CN1213368A - Hydroxamic acid based collagenase inhibitors - Google Patents

Abstract

Compounds of formula (I) wherein R and R<3> are each independently hydrogen, alkyl, alkenyl, alkynyl or aryl; R<1> is arylmethyl; and R<2> is alkyl, alkenyl, aryl, cycloalkyl or cycloalkenyl, and pharmaceutically acceptable derivatives thereof, are useful in the treatment and prophylaxis of disorders in which the overproduction of sCD23 is implicated.

Description

Collagenase inhibitors based on hydroxamic acid

The present invention relates to that the inhibitor of soluble human CD 23 forms new and treatment relevant with excessive Soluble CD23 (s-CD23) generation such as the purposes in autoimmune disease and the allergy illness.

CD23 (low nucleophilic power IgE acceptor FceR II, Blast2) for expressing the 45kDa II type integral protein on mature cell mutation surface, described cell comprises B and T lymphocyte, macrophage, NK, langerhans' cells, monocyte and blood platelet (Delespesse etc., Adv Immunol, 49[1991] 149-191). On acidophic cell, also there is CD23 sample molecule (Grangette etc., J Immunol, 143[1989] 3580-3588). CD23 is relevant with immunoreactive regulating action (Delespesse etc., Immunol Rev, 125[1992] 77-79) also. Human CD 23 exists with isomeric form-a and the b of two kinds of different adjustment, and its difference is on intracellular N end amino acid (Cell such as Yokota, 55[198] 6 11-618) only. In the people, form a isomers and only find at the B-lymphocyte, and the b type thing of being induced by IL 4 all is found in all can express the cell of CD23.

Known complete Cell binding CD23 (i-CD23) causes forming many well-defined solubility fragments (s-CD23) through the cracking by cell surface, these fragments are that the serial proteolysis by complexity produces, its mechanism it be unclear that (the J Biol Chem such as Bourget, 269[1994] 6927-6930). Although not yet confirm, but these proteoclastic main solubility fragments (Mr37,33, the 29 and 25KDa) order that can infer the terminal lectins domain of the common C of whole reservations and i-CD23 produces (the J Exp Med such as Letellier, 172[1990] 693-700) through the initial formation of 37kDa fragment. The mode of division is created in the stable 16kDa fragment that the C stub area is different from i-CD23 (the Eur J Immu nol such as Grenier-Brosette, 22[1992] 1573-1577) in the another kind of cell.

Several activity are attributed to people's film in conjunction with iCD23, have shown that they work in IgE regulates. Concrete activity comprises: a) antigen presentation, b) cytotoxicity of the acidophic cell of IgE mediation, c) the B cell is got back to the generation center d of lymph node and spleen) synthetic downward adjusting (the downregulation) (Delespesse etc. of IgE, Adv Immunol, 49, [1991] 149-191). As if three larger molecular weight Soluble CD23 fragments (Mr37,33 and 29kDa) have multifunctional cytokine character (playing a major role) in IgE produces. Therefore, the excessive generation of s-CD23 is relevant with the excessive generation of IgE, is for example extrinsic asthma, rhinitis, allergic conjunctivitis, eczema, atopic dermatitis and hypersensitive proof (Sutton and Gould, Nature of anaphylactia, 366, [1993] 421-428).

Other biologically active that comes from s-CD23 comprises the stimulation of B Growth of Cells and inducing of monocyte medium release. Like this, observed (the Blood such as Sarfati the patient who suffers from the B chronic lymphocytic leukemia, 71[1988] 94-98) and at the patient (Chomarat etc. that suffer from rheumatic arthritis, Arthritis and Rheumatism, 36 1993] rising of s-CD23 concentration in serum 234-242). Many document suggestion CD23 work in inflammation. At first, have been reported sCD23 and be incorporated into the extracellular acceptor, described acceptor is relevant with cell-mediated inflammatory reaction when activating. Thereby there are the direct activated mononuclear cell of report sCD23 TNF, IL-1 and IL-6 to discharge (Armant etc., the 108th volume, J.Exp.Med 105-1011 (1994)). It is reported that CD23 and B2-integrin adhesion molecule, CD11b and CD11c are at monocyte/macrophage interaction (S.Lecoanet-Henchoz etc., Immunity, 3 volumes; 119-125 (1995)), cause NO2, hydrogen peroxide and cell factor (IL-1, IL-6 and TNF) discharge. At last, IL-4 or IFN induce the expression of CD23 and are discharged by the person monocytic cell as sCD23. Film activates cAMP in conjunction with sCD23 acceptor and being connected of the anti-IgE immune complex of IgE/ or anti-CD23 mAb and IL-6 generates and thromboxane B2 generates, and proves the receptor-mediated effect of CD23 in inflammation.

Because these multiple character of CD23, suppress the compound that s-CD23 generates and just should have double action: a) by keeping i-CD23 to strengthen the synthetic negative feedback inhibition of IgE in the concentration of B cell surface, and b) the immunostimulatory cell factor active of the macromolecule solubility fragment of inhibition s-CD23 (Mr 37,33 and 29kDa). In addition, the inhibition of CD23 cracking can weaken monocytic activity that sCD23 induces and the generation of medium, and then the reaction that reduces inflammation.

The compound that international patent application no PCT/EP95/02693 (Smithkline Beechanm plc) openly suppresses matrix metalloproteinase (for example clostridiopetidase A, stromelysin and gelatinase) effect is the establishment agent that transfection enters the human soluble CD23 release of cells of mamma animals cultivating system. Known NMPI comprises the compound of the patent application description of listing in the table.

Table

Patent application	Disclosed compound	Specific compound and preparation method embodiment number
			US-A-4,595,700	Such as formula (I) compound of claim (1) definition, choose further in this manual auxiliary definition wantonly.	1 to 8.
US-A-4,599,361	1 to 7.
		GB-A-2 268 934	1 to 10.
GB-A-2 272 441	1 to 5.
		EP-A-0 231 081	1 to 8.
EP-A-0 236 872	1 to 28.
		EP-A-0 262 053	1 to 15.
EP-A-0 273 689	1 to 38.
		EP-A-0 276 436	1 to 44.
EP-A-0 274 453	1 to 8.
		EP-A-0 320 118	1 to 5.
EP-A-0 489 577	1 to 25.
		EP-A-0 489 579	1 to 4.
EP-A-0 497 192	1 to 80.
		EP-A-0 498 665	1 to 27.
EP-A-0 520 573	1 to 34.
		EP-A-0 574 758	1 to 43.
EP-A-0 575 844	1 to 27.
		EP-A-0 606 046	1 to 32.
EP-A-0 613 883	1 to 7.
		EP-A-0 621 270	1 to 40.
WO 90/05716	1 to 38.
		WO 90/05719	1 to 26.
WO 91/02716	1 to 17.
		WO 92/09563	Compound formula (1) or (2) such as claim (1) or (2) definition		1 to 21.

Table is continuous

Patent application	Disclosed compound	Specific compound and preparation method embodiment number
			WO 92/13831	Such as formula (I) compound of claim (1) definition, choose further in this manual auxiliary definition wantonly.	1 to 27.
WO 92/21360	1 to 5.
		WO 92/22523	I is to X.
WO 93/14096	1 to 8.
		WO 93/20047	1 to 14.
WO 93/24475	1 to 6.
		WO 93/24449	1 to 8.
WO 94/00119	1 to 86.
		WO 94/07481	1 to 15.
WO 94/12169	1 to 24.
		WO 94/21625	1 to 7.
WO 94/21612	1 to 116.
		WO 94/24140	1 to 5.
WO 94/25434	1 to 7.
		WO 94/25435	Embodiment 1
WO 95/04033	Embodiment 1 to 7
		WO 95/04715	Whole embodiment
WO 95/12603	Whole embodiment

According to the invention provides formula (I) compound:

Wherein R and R³Independent respectively is hydrogen, alkyl, alkenyl, alkynyl or aryl; R¹Be arylmethyl; And R²Be alkyl, alkenyl, aryl, cycloalkyl or cycloalkenyl group.

Alkyl, alkenyl and alkynyl refer to comprise straight chain and the branched group and optional by one or more aryl, heterocyclic radical, (C of being selected from that contains 6 carbon atoms of as many as at this_1-6) alkylthio group, (C_1-6) alkenyl thio, (C_1-6) alkynyl sulfenyl, arylthio, heterocycle sulfenyl, (C_1-6) alkoxyl, aryl (C_1-6) alkoxyl, aryl (C_1-6) alkylthio group, amino, single or two (C_1-6) group of alkylamino, cycloalkyl, cycloalkenyl group, carboxyl and its ester, hydroxyl and halogen replaces.

Cycloalkyl and cycloalkenyl group refer to comprise the group with 3-8 unit ring carbon atom and choose wantonly by above-mentioned group for alkyl, alkenyl and alkynyl at this and replace.

The term " aryl " that is used for herein comprises phenyl and naphthyl such as 2-naphthyl. The suitable aromatic yl group that comprises phenyl and naphthyl can be chosen wantonly by 5 of as many as, and 3 substituting groups of preferred as many as replace. Suitable substituting group comprises halogen, (C_1-6) alkyl, aryl (C_1-6) alkyl, (C_1-6) alkoxyl, (C_1-6) alkoxyl (C_1-6) alkyl, halo (C_1-6) alkyl, hydroxyl, nitro, amino, single and two-N-(C_1-6) alkyl amino, amide groups, acyloxy, carboxyl, carboxylate, carboxylate, carbamyl, single and two-N-(C_1-6) alkylcarbamoyl group, (C_1-6) alkoxy carbonyl group, aryloxy carbonyl, urea groups, guanidine radicals, sulfoamido, amino-sulfonyl, (C_1-6) alkylthio group, (C_1-6) alkyl sulphinyl, (C_1-6) alkyl sulphonyl, heterocyclic radical and heterocyclic radical (C_1-6) alkyl. Two the adjacent ring carbon atom can be by (C in addition_3-5) alkylidene chain is connected to form carbocyclic ring.

Be used for herein term " heterocyclic radical " and " heterocycle " unless otherwise indicated, be suitable for comprising fragrance with non-aromatic, monocycle and condense, described ring is suitable for containing four hetero atoms that are selected from oxygen, nitrogen and sulphur of as many as, and each ring can not replace or replaced by three substituting groups of as many as for example. The ring of each heterocycle is suitable for having 4-7, preferred 5 or 6 annular atomses. The ring system of the heterocycle that condenses can comprise the ring of carbocyclic ring and need to comprise the only ring of a heterocycle.

The substituting group of preferred heterocyclic group is selected from halogen, (C_1-6) alkyl, aryl (C_1-6) alkyl, (C_1-6) alkoxyl, (C_1-6) alkoxyl (C_1-6) alkyl, halo (C_1-6) alkyl, hydroxyl, amino, single and two-N-(C_1-6) alkyl amino, amide groups, carboxylate, carboxylate, carbamyl, single and two-N-(C_1-6) alkyl-carbonyl, aryloxy carbonyl, (C_1-6) alkoxy carbonyl group (C_1-6) alkyl, aryl, oxygen groups, urea groups, guanidine radicals, sulfoamido, amino-sulfonyl, (C_1-6) alkylthio group, (C_1-6) alkyl sulphinyl, (C_1-6) alkyl sulphonyl, heterocyclic radical and heterocyclic radical (C_1-6) alkyl.

Of the present invention special aspect, R is hydrogen or methyl, optional is replaced by arylthio or heterocyclic radical sulphur; And/or R¹Be benzyl group; And/or R²Be benzyl group; And/or R³Be hydrogen, methyl or benzyl.

According to other aspect, the invention provides formula (I) compound in illness that production for treating or prevention comprise the excessive generation of the s-CD23 purposes in the medicine of irritated, inflammation imbalance and autoimmune disease for example.

Another aspect of the present invention provides for example method of irritated, inflammation imbalance and autoimmune disease of illness that treatment or prevention comprise the excessive generation of s-CD23, and the method comprises to the people of needs or inhuman mammal with formula (I) compound.

The present invention also provide illness that treatment or prevention comprise the excessive generation of s-CD23 for example irritated, inflammation imbalance and autoimmune disease Pharmaceutical composition, said composition comprises formula (I) compound and optional pharmaceutically acceptable carrier.

Concrete inflammatory disease comprises CNS disorder for example Alzheimer's, multiple sclerosis and Multi-infarct dementia, and the sequelae of inflammation mediated apoplexy and brain trauma.

The pharmaceutically acceptable salt, solvate and the pharmaceutically acceptable derivates thereof that should be appreciated that formula (I) compound are also included among the present invention.

The salt of formula (I) compound for example comprises derived from inorganic or organic acid for example hydrochloric acid, hydrobromic acid, hydroiodic acid, p-toluenesulfonic acid, phosphoric acid, sulfuric acid, acetic acid, trifluoro formic acid, propionic acid, citric acid, maleic acid, fumaric acid, malonic acid, butanedioic acid, lactic acid, oxalic acid, tartaric acid and benzoic acid-addition salts.

Salt also available bases generates. This type of salt comprises that by salt inorganic or that organic base is derived for example alkali metal salt such as sodium or sylvite reach organic amine salt for example morpholine, piperidines, dimethylamine or diethyl amine salt.

Be surprisingly found out that the compounds of this invention is the potent optionally CD23 function inhibitor that reaches, and relatively shows the collagen enzyme inhibition activity of attenuating simultaneously with the compound of above-mentioned prior art.

The compounds of this invention can with any suitable conventional method preparation, for example use the method that is similar to following patent Introduction: WO 90/05716, WO 93/24475, WO 94/21625, WO 95/19956, WO 90/05719, WO 91/02716, WO 92/13831, WO 93/20047, EP-A-0214639, EP-A-0236872, EP-A-0274453, EP-A-0489577, EP-A-0489579, EP-A-0497192, EP-A-0574758 and USP 4599361.

Provide according to another aspect of the present invention to prepare the as defined above method of formula (I) compound, the method comprises:

(a) formula (II) compound deprotection:R-R wherein³As above definition, and X is the protecting group such as benzyl or TMS, or

(b) formula (III) compound:

R-R wherein³As defined above, with azanol or its reactant salt or

(c) formula (IV) compound:

R wherein¹-R ³As defined above, obtain formula (I) compound with thiol reactant, wherein R is the methyl that is replaced by alkylthio group, arylthio, aromatic alkylthio or heterocyclic radical sulfenyl, or

(d) formula (I) compound is transformed into the as defined above different compound of formula (I).

Formula (II), (III) and (IV) compound are new and form another aspect of the present invention.

Formula (II) but the azanol reaction of compound through type (III) compound and protection make. Formula (III) but compound through type (V) compound:

R-R wherein³As above definition, and Y makes for the hydrolysis such as the protecting group of t-butyl.

Suitable hydroxamic acid protecting group is known in this area, comprises benzyl, TMS, t-butyl and t-butyl dimethylsilyl.

Suitable carboxylic acid protecting group in this area be know and comprise t-butyl, benzyl and methyl.

Formula (V) but compound through type (VI) compound:Wherein, R¹-R ³Reach Y and as above define, and Z is for making ZCH₂-be the reduction preparation of the group of R.

Formula (V) but also through type (VII) compound of compound:

Wherein R, R1 and Y as defined above, and the derivative of formula (VIII) compound or its activation:

Wherein R2 and R3 as defined above, reaction makes.

Formula (III) compound also can be by formula (IX) compound:

R wherein¹-R ³As defined above, obtain formula (III) compound with thiol reactant, wherein R prepares for the methyl that is replaced by alkylthio group, arylthio, aromatic alkylthio or heterocyclic radical sulfenyl.

Formula (IX) compound also can be by formula (X) compound:

R wherein¹-R ³And the hydrolysis as defined above of Y makes.

Formula (X) but also through type (XI) compound of compound:

Wherein R1 and Y make with the reaction of formula (VIII) compound or derivatives thereof as defined above as defined above.

Described raw material and other reagent can by commerce buy or can by know and conventional method synthetic.

The isomers of the compounds of this invention comprises that stereoisomer can be made into the mixture of this type of isomers or independent isomers. Described independent isomers can be by the preparation of arbitrary suitable method, and for example described independent stereoisomer can be initial through the single-minded chemical synthesis preparation of solid or by using known method to make through separating non-enantiomer mixture by chiral substrates. Aspect preferred, the invention provides (I A) compound:

Preferred described compound separates with basically pure form.

Soluble human CD 23 formation inhibitor as described herein has useful medical character. Preferred described reactive compound is with pharmaceutically acceptable composition administration.

The administration of described composition preferred oral. Yet also can adopt other administering mode, for example with spray, aerosol form or other conventional inhalation method treatment breathing problem; Or to suffering from patient's gastrointestinal tract external administration in heart failure. Other alternative form of medication comprises sublingual administration or cutaneous penetration.

Described composition can be tablet, capsule, powder agent, granule, lozenge, suppository, reproducible powder agent, or liquid preparation, for example the form of the outer solution of oral or aseptic intestines and stomach or suspension.

Be unit dosage form in order to obtain the preferred present composition of consistent administration.

The unit dose formulations form of oral administration can be tablet and capsule and can contain conventional assistant such as adhesive, for example syrup, Arabic gum, gelatin, sorbierite, tragacanth or polyvinylpyrrolidone; Filler, for example lactose, sugar, cornstarch, calcium phosphate, sorbierite or glycerine; Film-making lubricant, for example dolomol; Disintegrant, for example starch, polyvinylpyrrolidone, hydroxyl amylcose acetate sodium or microcrystalline cellulose; Or pharmaceutically acceptable wetting agent such as SDS.

Described solid composite can make by the conventional method of mixing, filling or film-making. Repeating married operation can be used for active agent is scattered in those compositions of using a large amount of fillers. It naturally is conventional that this kind operates in this area. Described tablet can be according to the method dressing of knowing in conventional pharmaceutical practice, and is particularly enteric coated.

Oral liquid can be for example emulsion, syrup or elixir form, or can be used as the mummification compound that water before use or other suitable solvent copy and provide. This kind liquid preparation can contain conventional additives such as suspending agent, for example sorbierite, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel, hydrogenation edible fat; Emulsifying agent, for example lecithin, Arlacel-80 or Arabic gum; Non-water-soluble matchmaker (can comprise edible oil), for example apricot kernel oil, fractionated coconut oil, grease such as glyceride, propane diols or ethylene glycol; Anticorrisive agent, for example methyl p-hydroxybenzoate or propyl ester or sorbic acid; If necessary, also can contain conventional flavor enhancement or colouring agent.

For the intestines and stomach external administration, the liquid unit doses form is by using described compound and aseptic solvent makes, and depend on used concentration can or for suspend or be dissolved in the solvent. Described compound dissolves in water for injection and filtration sterilization in pack into suitable bottle or ampoule and before the sealing during preparation injection solution. More advantageously assistant such as local anesthetic, anticorrisive agent and buffer can be dissolved in the described solvent. In order to strengthen stability, described composition after adding described bottle freezing and vacuum except anhydrating. The outer suspending agent of intestines and stomach is suspended in the solvent except described compound rather than is dissolved in wherein, and sterilization can not be accomplished by filtration and substantially prepares by identical mode outward. Described compound can be exposed in the oxirane before in being suspended in aseptic solvent sterilizes. More advantageously, surfactant or wetting agent are included in the described composition so that described compound is easy to even distribution.

The compounds of this invention also is suitable for as the snuffing agent of respiratory tract administration or aerosol or as the solution of sprayer, or as the fine pulvis of inhalation, and being combined separately or with inert carrier such as lactose provides. In such cases, the particle of reactive compound is suitable for having diameter less than 50 microns, preferably less than 10 microns for example diameter in the scope of 1-50 micron, 1-10 micron or 1-5 micron. If suitably, can comprise a small amount of other anti-asthmatic agent and bronchodilator, for example sympathomimetic amine such as isoprel, dilabron, broncovaleas, phyenlephrinium and ephedrine; Xanthine derivative such as theophylline and aminophylline and corticosteroid such as prednisolone and adrenal gland excitant such as ACTH.

Can contain 0.1%-99% (weight), the active material of preferred 10-60% (weight) according to the described composition of the method for administration. The scope of preferred inhalation is 10-99%, 60-99% particularly preferably, for example 90,95 or 99%.

The attritive powder preparation can by dosing or by suitable suction activating apparatus with aerosol drug delivery.

Suitable dosing aerosol formulation comprises conventional propellant, cosolvent such as ethanol, surfactant such as oleyl alcohol, lubricant such as oleyl alcohol, drier such as calcium sulfate and density adjuster such as sodium chloride.

Suitable sprayer solution is to wait to ooze sterile solution, optional buffering for example between pH4-7, contain as many as 20mg/ml compound (but more generally containing 0.1-10mg/ml), be used for the standard spraying apparatus and use.

Effective dose depends on the compounds of this invention relative effectivenes, the order of severity for the treatment of disease and patient's body weight. The UD of the suitable present composition can comprise 0.1-1000mg the compounds of this invention (being 0.001-10mg through sucking) and more generally contain 1-500mg, for example 1-25 or 5-500mg. This kind composition can be by to dosage administration every day of the per day for adults 1mg-1g of 70kg and particularly 5-500mg 1-6 time, is more typically every day 2-4 time. Namely about 1.4 * 10^-2In mg/kg/sky-14mg/kg/ days the scope and particularly 7 * 10^-2In the scope of mg/kg/ days-7mg/kg/ days.

Following embodiment illustrates the present invention but does not limit in any form it. Biological test method method 1: use following method research test-compound to suppress the ability that Soluble CD23 discharges. RPMI 8866 cell membrane CD23 lytic activities are analyzed:

Express the B clone of the people Epstein-Barr virus Transformation of high concentration CD23, and the plasma membrane of RPMI 8866 cells (Sarfati etc., Immunology 60[1987] 539-547) usefulness aqueous solution extraction method purifying. The N of cell in the Parr pressure pan of Eddy diffusion in homogenate buffer (20mM HEPES pH7.4,150mMNaCl, 1.5mM MgCl2,1mM DDT)₂Cavitation is smashed, and the plasma membrane that mixes with other film part is by centrifugal recovery under 10000Xg. Light precipitation is suspended in 0.2M potassium phosphate (pH7.2, every 1-3g wet cell uses 2ml) and discards the nuclear precipitation. Film further separates (in 0.25M sucrose between (ref) through being allocated in Dextran500 (6.4%w/w) and polyethylene glycol (PEG) 5000 (6.4%w/w), the amount of every 10-15mg LMP-1 6g) [Morre and Morre, Bio Techniques 7,946-957 (1989)]. Separate and collect PEG (top) phase through simply centrifugal (1000Xg), with 20mM potassium phosphate buffer agent (pH7.4) dilution 3-5 doubly, and under 100000Xg this film in mutually of centrifugal recovery. Be suspended in precipitation in the PBS and consist of enrichment 3-4 doubly plasma membrane and other cell membrane (for example, lysosome, Golgi). With gained film five equilibrium and be stored in-80 ℃. Partly obtain the plasma membrane of 10 times of enrichments at 6.6%Dextran/PEG.

The film that separates is hatched in 37 ℃ and is reached 4 hours and produce the CD23 fragment, and this fragment is got by 0.2 micron Durapore filter (Millipore) filtration preparing 1 cancellation this analysis with the 5uM that derives from P30994 by described film. The sCD23 EIA kit (Birmingham that derives from The Binding Site by described film release, UK) mensuration or the anti-CD23 mAb[Rowe of similar application MHM6 etc., Int.J.Cancer, 29,373-382 (1982)] or the capture antibody of another anti-CD23 mAb conduct in the EIA interlayer. The amount of the Soluble CD23 that the 0.5ug memebrane protein in cumulative volume 50ul PBS makes is recorded by EIA, and with the amount of gained in the presence of various control of the concentration agent relatively. Inhibitor is preparation in the aqueous solution or dimethyl sulfoxide (DMSO) (DMSO), and the concentration of final DMSO is no more than 2%. Measure the IC50 value by 50% concentration curve that suppresses that the sCD23 that is observed by the difference of the sCD23 of hatching with respect to the contrast of without inhibitor generates. Method 2: use following method research test-compound to suppress the ability of clostridiopetidase A. The clostridiopetidase A inhibition analysis:

Effectiveness as the collagenase inhibitors compound is measured (Anal.Biochem.99 by Cawston and Barrent method, 340-345,1979, be incorporated herein by reference), the method comprises the 1mM solution of testing inhibitor or its dilution and being expressed and collagen and people's recombinant collagen enzyme of purifying (cushion with 150mMTris by E.Coli. through the synovia fibroblast cloning, pH 7.6, contain 15mM calcium chloride, 0.05%Brij35,200mM sodium chloride and 0.02% Sodium azide) hatched 18 hours in 37 ℃. Described collagen is through the acidylate of Cawston and Murphy method (at Enzymology 80,711, the method in 1981) preparation³H 1 type bovine collagen. Described sample centrifugation goes out not digest collagen, and tells the mensuration that equal portions radioactivity supernatant is hydrolyzed at scintillation counter. With described clostridiopetidase A in the presence of 1mM inhibitor or its dilution activity and the specific activity of unrestraint agent contrast, and with acquired results with to the effective concentration (IC of 50% clostridiopetidase A₅₀) provide. Embodiment 1 N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-benzyl acid amidesA) 2-benzal-4-tert-butoxy butanedioic acid

The solution of the methyl alcohol (20ml) of 2-benzal-4-tert-butoxy methyl succinate (2g, 7.25mmol) is processed with sodium hydroxide solution (2M, 7.25ml, 14.5mmol), and this mixture was stirred 16 hours in 20 ℃. The described reactant liquor of Vacuum Concentration also adds entry (20ml). The aqueous solution extracted with diethyl ether (2 * 20ml), extract again (3 * 25ml) with the 2M hcl acidifying and with ether. Dry (MgSO₄) the ether layer of this acidizing extraction thing, filter and evaporation obtains white solid acid (1.74g, 92%). Mp110-115 ℃. d_H(CDCl ₃) 1.46 (9H, s), 3.47 (2H, s), 7.38 (5H, m), 7.94 (1H, s), 12.58, brs). b) N-(2-benzal-4-tert-butoxy succinyl group)-(S)-phenylalanine-N '-benzyl acid amides

In 0 ℃ with 2-benzal-4-tert-butoxy butanedioic acid (524mg, 2mmol), (S)-phenylalanine-N '-benzyl amide hydrochloride (580mg, 2mmol), I-hydroxybenzotriazole hydrate (486mg, 3.6mmol) and diisopropylethylamine (0.35ml, the solution of DMF 2mmol) (20ml) 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (424mg, 2.2mmol) process, and this reactant was stirred 1 hour and stirred 16 hours in 20 ℃ in 0 ℃. After the evaporation, residue distributes in saturated sodium bicarbonate (25ml) and carrene (50ml). Organic layer is successively used saturated sodium bicarbonate (2 * 25ml), 10% citric acid (2 * 25ml) and salt solution (25ml) washing and dry (MgSO₄). Filter and concentratedly obtain residue, obtain canescence foam-like title compound (850mg, 85%) through silica gel column chromatography (chloroform-methanol 100: 1). d_H(CDCl ₃) 1.40 9H, s), 3.25 (2H, m); (3.32 1H, d, J=16.5Hz), 3.50 (1H; d, J=16.5Hz), 4.40 (2H; m), 4.80 (1H, dd; J=6.9,7.2Hz), 6.75 (2H; m), 7.26 (16H, m). c) N-(2-(R/S)-benzyl-4-tert-butoxy succinyl group)-(S)-phenylalanine-N '-benzyl acid amides

With the hydrogenation 4.5 hours under atmospheric pressure of the solution of the ethanol (50ml) of N-(2-benzal-4-tert-butoxy succinyl group)-(S)-phenylalanine-N '-benzyl acid amides (790mg, 1.58mmol). Filter described reactant, reduction vaporization also obtains residue mp 95-100 ℃ of the desired substance (660mg, 83%) of 1: 1 mixture of white solid and diastereoisomer through silica gel column chromatography (chloroform-methanol 100: 1). d_H(CDCl ₃) 1.25+1.41 (9H; 2xs); (2.3-3.5 7H, m), 4.05-4.70 (2H; m); (4.60-4.80 1H, m), 5.33-6.02 (2H; m), 6.7-7.4 (15H). d) N-(2-(R/S)-benzyl-4-hydroxyl succinyl group)-(S)-phenylalanine-N '-benzyl acid amides

With N-(2-(R/S)-benzyl-4-tert-butoxy succinyl group)-(S)-phenylalanine-N '-benzyl acid amides (650mg; 1.3mmol) carrene (15ml) solution process with trifluoroacetic acid (15ml), and stirred 4 hours in 20 ℃. With the evaporation of this reactant and use the toluene coevaporation. Grind mp172-5 ℃ of the acid (470mg, 81%) that residue obtains 1: 1 mixture of white solid and diastereoisomer with ether. d_H[(CD ₃) ₂SO]2.01-3.03(7H,m),[4.22(d,J=5.8Hz)+4.27(d,J=5.4Hz),total 2H],4.53 (1H,m),7.02-7.31(15H,m),[8.18(t,J=5.8Hz)+8.27(t,J=5.9Hz,total 1H],[8.22(d,J=8.3Hz) +8.35(d,J=8.5Hz),total 1H],12.1(1H,brs,CO ₂H). e) N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-benzyl acid amides

To be 1: 1 diastereoisomer N-(2-(R/S)-benzyl-4-tert-butoxy succinyl group)-(S)-phenylalanine-N '-benzyl acid amides (250mg; 0.56mmol), diisopropylethylamine (0.2ml; 1.12mmol) and O-TMS azanol (300mg; 2.8mmol) carrene (10ml) solution be chilled to 0 ℃; and under argon gas, process with bromo-tripyrrole alkane (pyrrolidino) phosphorus hexafluorophosphate (PyBroP) (160mg, 0.67mmol). This reactant was stirred 1 hour and stirred 16 hours in 20 ℃ in 0 ℃. With organic layer with the salt water washing (2 * 10ml), dry (MgSO₄) and flash to sticky solid. This residue uses VYDAC albumen and peptide C 18 reversed-phase columns through preparation property HPLC chromatography, with the H of 0.1%TFA₂O: the 0.1%TFA-H of 70% acetonitrile₂1: 1 mixed liquor of O is with the flow velocity wash-out of 20ml/min. At first the diastereoisomer of wash-out (9.7min) proves mp195-7 ℃ of N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-benzyl acid amides (50mg, 19%). d_H[(CD ₃) ₂SO] 1.88 (1H, dd, J=6.9; 14.9Hz), 2.09 (1H, dd; J=7.5,14.9Hz), 2.72-3.05 (5H; m); (4.21 2H, m), 4.47 (1H; m); (7.23 15H, m), 8.18 (2H; m); (8.70 1H, brs), 10.35 (1H; brs). the diastereoisomer (12.5min) of the second washing proves mp203-5 ℃ of N-(2-(S)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-benzyl acid amides (30mg, 12%).

d _H[(CD ₃) ₂SO] 1.92 (1H, dd, J=6.1; 15.1Hz), 2.23 (1H, dd; J=8.8,15.0Hz), 2.35-2.75 (3H; m), 2.98 (2H, m); (4.27 2H, m), 4.45 (1H; m), 7.21 (15H, m); (8.33 1H, d, J=8.5Hz); 8.45 (1H; m), 8.71 (1H, s); (10.42 1H, s). embodiment 2 N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea

A) 4-(S)-benzyl-3-(3-phenyl propiono)-2-oxazolidone

In-65 ℃ with n-BuLi (1.6M (in hexane) 101ml; 178mmol) drop in oxolane (250ml) solution of 4-(S)-benzyl-3-(3-phenyl propiono)-2-oxazolidone (23.9g, 135mmol). The orange solution of gained was stirred 30 minutes, then be added dropwise to oxolane (40ml) solution of 3-phenyl propionyl chloride (25g, 148mmol). 1.5 after hour, this reactant is poured in the ice-cooled 2M hydrochloric acid (200ml), and with ethyl acetate extraction (3 * 150ml). The organic extract that merges washs with 2M hydrochloric acid (150ml), sodium bicarbonate solution (150ml) and salt solution (100ml). Dry (MgSO₄) and the evaporation after, residue is obtained the white needles title compound by crystallization in the ether. Mp109-111 ℃ of .d_H(CDCl ₃) 2.75 (1H, dd, J=13.5,10Hz), 2.96-3.07 (2H, m); (3.17-3.38 3H, m), 4.14 (1H, s); (4.16 1H, d, J=2), 4.60-4.70 (1H; m), 7.15-7.35 (10H, m). b) 4-(S)-benzyl-3-(2-tert-butoxycarbonyl methyl] 3-phenyl) propiono-2-oxazolidone

In-20 ℃ n-BuLi (1.6M (in hexane) 28.3ml, 45mmol) is dropped to 1,1,1-3,3, in the oxolane of 3-HMDS (9.6ml, 45mmol) (60ml) solution. After 45 minutes this solution is chilled to-75 ℃, and is added dropwise to oxolane (60ml) solution of 4-(S)-benzyl-3-(3-phenyl propiono)-2-Evil oxazolidone (10g, 32mmol). Stir this solution 1 hour, and then be warmed to-50 ℃ with 1 hour. Be chilled to oxolane (60ml) solution that is added dropwise to the monobromo-acetic acid tert-butyl ester (7.8ml, 48mmol) after-75 ℃, and stir this solution and then be warmed to-20 ℃ with 2.5 hours in 1 hour. This reactant liquor is poured in the saturated ammonium chloride solution (100ml), and with extracted with diethyl ether (3 * 10ml). The organic extract that merges washs with 2M hydrochloric acid (50ml), sodium bicarbonate solution (50ml) and salt solution (50ml). Dry (MgSO₄) and evaporation after obtain white solid title compound (12.3g, 90%) with ether/hexane (1: 4) grinding gained yellow oil.

mp111.5-112.5℃.d _H(CDCl ₃) 1.4 (9H, s), 2.37 (1H, dd, J=17,4Hz), 2.64 (1H, dd, J=13,9Hz), 2.72 (1H, dd, J=14,10Hz), 2.85 (1H, dd, J=17,11Hz), 3.01 (1H, dd, J=13.6Hz), 3.31 (1H, dd, J=13.5,3Hz), 3.93 (1H, t, J=6Hz), 4.07 (1H, dd, J=9,2.5Hz), 4.44-4.57 (2H, m), 7.18-7.37 (10H, m). c) 2-(R)-benzyl-4-tert-butoxy butanedioic acid

In 0 ℃ with 27.5% hydrogen peroxide (9.65ml; 78mmol) process 4-(S)-benzyl-3-(2-tert-butoxycarbonyl methyl]-3-phenyl) propiono-2-oxazolidone (5.5g; 13mmol) the solution in 3: 1 THF/ water (200ml); and before water (100ml) solution that adds lithium hydroxide (1.1g, 26mmol), stirred 10 minutes. Stir this reactant liquor 30 minutes in 0 ℃, and destroy excessive peroxide by water (58ml) solution that adds 1.5M sodium sulfite (11g, 87mmol). This solution with sodium bicarbonate buffer to pH9 and evaporate THF. Residue distributes in water (300ml) and carrene (300ml), and (2 * 200ml) wash water layers with carrene. This water layer is with the 1MHCl acidifying and use ethyl acetate extraction (3 * 200ml). Dry organic layer (MgSO₄), filtration and evaporation obtain the acid (3.4g, 100%) of required clarification grease. d_H(CDCl ₃) 1.42 (9H, s), 2.35 (1H, dd, J=4.7,16.8Hz), 2.56 (1H, dd, J=8.7,16.8Hz), 2.77 (1H, dd, J=10.4,15.4Hz), 3.09 (2H, m), 7.17-7.34 (5H, m), sour proton is not seen. D) 2-(R)-benzyl-4-tert-butoxy succinyl group-(S)-phenylalanine-N '-methyl nitrosourea

With 2-(R)-benzyl-4-tert-butoxy butanedioic acid (1g, 3.78mmol), (S)-phenylalanine-N '-methyl nitrosourea hydrochloride (812mg, 3.78mmol), N, N-diisopropylethylamine (0.66ml, 3.78mmol), I-hydroxybenzotriazole hydrate (919mg, 6.8mmol) and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (797mg, 4.16mmol) DMF solution such as embodiment 1b) process, obtain white solid title compound (1.15g, 72%) via ethyl acetate-hexane recrystallization. d_H(CDCl ₃) 1.43 (9H, s), 2.38 (1H, dd; J=4.4,16.8Hz), 2.57 (3H, d; J=4.7Hz), 2.53-2.99 (5H, m) 3.15 (1H, dd; J=5.8,13.8Hz), 4.54 (1H, m); (5.33 1H, br m), 5.90 (1H, br d; J=8Hz), 7.12-7.31 (10H, m). e) N-(2-(R)-benzyl-4-hydroxyl succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea

With 2-(R)-benzyl-4-tert-butoxy succinyl group-(S)-phenylalanine-N '-methyl nitrosourea (1.02g; 2.41mmol) and carrene (10ml) solution of TFA (10ml) according to embodiment 1d) process the required acid (830mg, 94%) that obtains white solid. d_H[(CD ₃) ₂SO] 2.03 (1H, dd, J=5.5; 16.5Hz), 2.33 (1H, dd; J=8.4,16.6Hz), 2.50 (3H; d, J=4.6Hz), 2.56 (1H; m), 2.75-2.99 (4H, m); (4.39 1H, m), 7.14-7.27 (10H; m); (7.47 1H, m), 8.17 (1H; d; J=8.2Hz), 12.07 (1H, brs). f) N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea

With N-(2-(R)-benzyl-4-hydroxyl succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea (0.8g; 2.17mmol) THF (50ml) solution be chilled to-20 ℃; and with N-methylmorpholine (0.29ml; 2.61mmol) and carbonochloridic acid isobutyl ester (0.34ml, 2.61mmol) is processed and this reactant liquor was stirred 1 hour in-20 ℃. Be added dropwise to O-TMS-azanol (2.2ml, 20.9mmol), this reactant liquor is warmed to room temperature and stirred 16 hours. Then, this reactant liquor evaporates and is allocated in ethyl acetate and 10% citric acid. Organic layer salt water washing and dry (MgSO₄). Filtration and evaporation obtain solid, and this solid obtains mp188-190 ℃ of white solid title compound (300mg, 36%) by ethyl acetate-recrystallizing methanol. d_H[(CD ₃) ₂SO] 1.91 (1H, dd, J=7.2; 15.0Hz), 2.06 (1H, dd; J=7.2,15.0Hz), 2.50 (4H; m), 2.75 (2H, m); (2.95 2H, m), 4.31 (1H; m), 7.06-7.27 (10H, m); (7.38 1H, m), 8.11 (1H; d; J=8.2Hz), 8.73 (1H, s); (10.4 1H, s). embodiment 3 N-(2-(R)-benzyl-4-azanol base-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-DL-Phenylalanine amide

A) 3-(R)-phenyl-lactic acid benzyl ester

THF solution triethylamine (23.5ml, 17.02g with 3-(R)-phenyl-lactic acid (20.381g, 123mmol), 168.5mmol) and bromo benzyl (16.2ml, 23.18g, 135.3mmol) process, and with the lower stirring of mixture backflow 1 hour. Cooled and filtered is removed precipitation and evaporated filtrate. Residue is dissolved in ethyl acetate, with saturated sodium bicarbonate, 1M HCl, salt water washing, and dry (MgSO₄). Filter and evaporate and obtain yellow oily title compound (24.086g, 76%). d_H[(CD ₃) ₂SO] 2.84 (1H, dd, J=7.97,13.75Hz), 2.97 (1H, dd.J=5.5,13.75Hz), 4.26-4.34 (1H, m), (5.09 2H, s), 5.62 (1H, d, J=6.32Hz), 7.16-7.43 (10H, m) b) 2-(R)-benzyl-3-tert-butoxycarbonyl Dibenzyl succinate

In 0 ℃ of DMF (165ml) solution that sodium hydride (60% is suspended in the mineral oil 3.37g, 84.3mmol) is added to the benzyl malonic acid tert-butyl ester (21.1g, 84.4mmol). This solution is warmed to room temperature and stirred 1 hour under argon gas.

In 0 ℃ with trifluoromethanesulfanhydride anhydride (21.85ml, 36.6g, 130.2mmol) in the solution of the disposable carrene (220ml) that adds to 3-(R)-phenyl-lactic acid benzyl ester (24g, 93.75mmol) and pyridine (7.09ml, 86.8mmol). This solution stirred under argon gas 1 hour. This mixture is with ice-cooled water, salt water washing, dry (MgSO₄) and filter.

In 0 ℃ of DMF solution that gained filtrate is added to malonate, and this solution is warmed to room temperature and stirred 16 hours. After the evaporation, residue is dissolved in ethyl acetate, with saturated sodium bicarbonate solution, 1M HCl, salt water washing and dry (MgSO₄). Filtration and evaporation obtain residue, and this residue obtains oily title compound (24.67g, 60%) through silica gel column chromatography (5% ethyl acetate-hexane). d_H(CDCl ₃) 1.35 (9H, d, J=11.55Hz) 2.84-3.02 (2H, m), 3.38-3.48 (1H, m), 3.68 (1H, dd, J=3.7,9.5Hz), 4.86-5.19 (4H, m), 7.03-7.37 (15H, m) c) 2-(R)-benzyl-4-tert-butoxy-3-vinyl butanedioic acid

Process isopropyl alcohol (90ml) solution of 2-(R)-benzyl-3-tert-butoxycarbonyl Dibenzyl succinate (14.64g, 30mmol) and in normal pressure hydrogenation with 10%Pd/C. Filter described reactant and process filtrate with piperidines (8.91ml, 90mmol) and formaldehyde (37%, 36.63ml, 450mmol), in stirred overnight at room temperature. After the evaporation residue is dissolved in the ethyl acetate, with 1M HCl, salt water washing and dry (MgSO₄). Filter and evaporate and obtain colorless oil title compound (7.17g, 87%). d_H(CDCl ₃) 1.48 (9H, s), 2.96 (1H, dd, J=8.25,14.02Hz), 3.26 (1H, dd, J=7.98,14.02Hz), 3.75 (1H, t, J=7.98Hz), 5.55 (1H, s), 6.23 (1H, s), 7.14-7.30 (5H, m), sour proton is not seen. D) N-(2-(R)-benzyl-4-tert-butoxy-3-vinyl succinyl group)-(S)-DL-Phenylalanine amide

In 0 ℃ with 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (5.35g, 27.96mmol) processing 2-(R)-benzyl-4-tert-butoxy-3-vinyl butanedioic acid (7.083g, 25.63mmol), (S)-DL-Phenylalanine amide (4.68g, 23.3mmol), diisopropylethylamine (3.97ml, 23.3mmol) and I-hydroxybenzotriazole hydrate (7.16g, 46.4mmol) the solution of DMF (80ml), and in 0 ℃ this reactant was stirred 1 hour, in stirring at room 16 hours. After the evaporation, residue is dissolved in the carrene, with 10% citric acid, saturated sodium bicarbonate washing and dry (MgSO₄). Filtration and evaporation obtain residue, and residue is obtained white solid title compound (5.83g, 60%) through silica gel column chromatography (7: 3 to 3: 7 gradient elutions of hexane-ethyl acetate). d_H[(CD ₃) ₂SO] 1.39 (9H, s), 2.68-2.79 (2H, m); (2.92-3.05 2H, m), 3.72 (1H, t; J=7.42Hz), 4.38-4.47 (1H, m); (5.44 1H, s), 5.94 (1H; s), 7.03-7.25 (12H, m); (7.90 1H, d, J=8.25Hz). e) N-(2-(R)-benzyl-4-hydroxyl-3-vinyl succinyl group)-(S)-DL-Phenylalanine amide

Process N-(2-(R)-benzyl-4-tert-butoxy-3-vinyl succinyl group)-(S)-DL-Phenylalanine amide (2.11g with trifluoroacetic acid (50ml), carrene 5mmol) (50ml) solution was in stirring at room 3 hours. Evaporate this reactant and use the toluene coevaporation. Residue grinds with ether and obtains white solid title compound (1.59g, 87%). d_H[(CD ₃) ₂SO] 2.70-2.78 (2H, m), 2.92-3.05 (2H, m); (3.77 1H, t, J=7.42Hz), 4.36-4.44 (1H; m), 5.48 (1H, s); (6.02 1H, s), 7.04-7.24 (12H; m), 7.87 (1H, d; J=8.53Hz), 12.59 (1H, br.s). f) N-(2-(R)-benzyl-4-hydroxyl-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-DL-Phenylalanine amide

Process N-(2-(R)-benzyl-4-hydroxyl-3-vinyl succinyl group)-(S)-DL-Phenylalanine amide (1.48g in room temperature with 2-mercapto-thiophene (5.07ml); 3.9mmol) methyl alcohol (20ml) solution, in the argon gas lower heating 18 hours that refluxes. Evaporate this reactant and use the toluene coevaporation. Residue grinds with ether and obtains white solid title compound (1.63g, 86%). d_H[(CD ₃) ₂SO] 1.95 (1H, dd, J=3.03; 12.92Hz), 2.28-2.75 (6H, m); 2.98 (1H, dd, J=4; 13.5 Hz), 4.32-4.41 (1H, m); (6.32 1H, s), 6.91-7.27 (13H; m), 7.60 (1H, dd; J=1.37; 5.22Hz), 8.19 (1H, d; J=8.53Hz); (12.70 1H, br, s) g) N-(2-(R)-benzyl-4-azanol base-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-DL-Phenylalanine amide

Under argon gas; at-20 ℃ with N-methylmorpholine (0.42ml; 3.6mmol) and carbonochloridic acid isobutyl ester (0.48ml; 489mg; 3.6mmol) processing N-(2-(R)-benzyl-4-hydroxyl-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(the S)-THF (25ml) of DL-Phenylalanine amide (1.445g, 3mmol) and solution of DMF (10ml). In-20 ℃ stir 1 hour after, process this solution by dripping O-TMS azanol (2.6ml, 2.53g, 21mmol), in stirring at room 16 hours. After the evaporation, residue is dissolved in ethyl acetate, with 10% citric acid, salt water washing and dry (MgSO₄). After the filtration, evaporation is also ground with ether, and heat solid and filtered residue in ethyl acetate (after repeating this operation for several times) obtain white solid title compound (25mg). d_H[(CD ₃) ₂SO] 1.81 (1H, dd, J=2.3; 12.5Hz), 2.25-2.66 (6H, m); 2.97 (1H, dd, J=3.7; 13.5 Hz), 4.25-4.33 (1H, m); (5.89 1H, s), 6.85 (1H; s), 6.96-7.24 (12H, m); 7.56 (1H, dd, J=1.2; 5.4Hz), 8.13 (1H, d; J=8.52Hz), 8.98 (1H, s); (10.71 1H, s). embodiment 4 N-(2-(R)-benzyl-4-azanol base-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea

A) N-(2-(R)-benzyl-4-hydroxyl-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea

Prepare according to embodiment 3f; but use N-(2-(R)-benzyl-4-hydroxyl-3-vinyl succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea (200mg instead; 0.53mmo1) obtain white solid title compound (100mg, 38%). d_H[(CD ₃) ₂SO] 1.94 (1H, dd, J=3.2; 12.8Hz), 2.28-2.72 (6H, m); (2.44 3H, d, J=4.7Hz); 2.93 (1H, dd, J=4.1; 13.5Hz), 4.37 (1H, m) .6.79 (1H; m), 7.01-7.27 (12H, m); 7.60 (1H, dd, J=1.5; 5.1Hz); (8.23 1H, d, J=8.8Hz); (12.7 1H, brs). b) N-(2-(R)-benzyl-4-azanol base-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea

Prepare white solid title compound (35%) according to embodiment 3g. d_H[(CD ₃) ₂SO]1.83(1H,d,J=10.5Hz),2.30(1H,m),2.39(3H,d,J=4.7Hz),2.50-2.63(5H,m), 2.94(1H,dd,J=4.1,13.7Hz),4.33(1H,m),6.45(1H,m),7.00(4H,m),7.17(8H,m),7.56 (1H,dd,J=1.4,5.2Hz),8.20(1H,d,J=8.2Hz),8.97(1H,brs),10.70(1H,brs).

Activity data Compound C D23 protease suppresses clostridiopetidase A and suppresses

                 ％(1uM)           IC ₅₀UM embodiment 3 105 0.93 embodiment 4 85 0.37 comparative examples^*         96                  0.005 ^*Comparative examples is the embodiment 2 among the WO 90/05719, following formula: compound:

Wherein R is CH₂S-(2-thienyl), R¹It is methyl.

Claims (10)

1. formula (I) compound or its pharmaceutically acceptable derivates, described formula (I) compound is as follows:

Wherein R and R³Independent respectively is hydrogen, alkyl, alkenyl, alkynyl or aryl; R¹Be arylmethyl; And R²Be alkyl, alkenyl, aryl, cycloalkyl or cycloalkenyl group.

2. formula (I A) compound or its pharmaceutically acceptable derivates, described formula (I A) compound is as follows:R to R wherein³As defining such as claim 1.

3. claim 1 or 2 compound, wherein R is hydrogen or methyl, is optionally replaced by arylthio or heterocycle sulfenyl; And/or R¹It is benzyl; And/or R²It is benzyl; And/or R³Hydrogen, methyl or benzyl.

4. be selected from following compound: N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-benzyl acid amides; N-(2-(R)-benzyl-4-azanol base succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea, N-(2-(R)-benzyl-4-azanol base-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-DL-Phenylalanine amide and N-(2-(R)-benzyl-4-azanol base-3-(S)-[2-thiophene sulphomethyl] succinyl group)-(S)-phenylalanine-N '-methyl nitrosourea.

5. be used for the treatment of or prevent to relate to purposes in the medicine of disease of the excessive generation of s-CD23 in production according to each compound of the claims.

6. be used for the treatment of or prevent to relate to the method for the disease of the excessive generation of s-CD23, the method comprises to the people of needs or non-human mammal clothes with each compound among the claim 1-4.

7. be used for the treatment of or prevent to relate to the composition of the disease of the excessive generation of s-CD23, said composition comprises each compound and optional pharmaceutically acceptable carrier of claim 1-4.

8. each the method for compound of preparation claim 1-4, the method comprises:

(a) formula (II) compound deprotection:R to R wherein³Such as claim 1 definition, X is the protecting group such as benzyl or TMS, or

(b) make formula (III) compound:

R to R wherein³Such as claim 1 definition and azanol or its reactant salt, or

(c) make formula (IV) compound:

R wherein¹-R ³Such as claim 1 definition, obtain formula (I) compound with thiol reactant, wherein R is the methyl that is replaced by alkylthio group, arylthio, aromatic alkylthio or heterocycle sulfenyl, or

(d) formula (I) compound is transformed into different formula (I) compound.

9. the formula defined such as claim 8 (II), (III) or (IV) compound.

10. formula (V) compound:

R-R wherein³Such as claim 1 definition, Y is the protecting group such as tert-butyl; Or formula (VI) compound:

Wherein, R¹-R ³And Y as defined above, and Z is for making ZCH₂It is the group of R; Or formula (IX) compound:

R wherein¹-R ³As defined above; Or formula (X) compound:

R wherein¹-R ³And Y as defined above.