CN105837479B - Hydrazine nitrile cathepsin K inhibitor and its application in treatment osteoarthritis drugs are prepared - Google Patents

Abstract

Hydrazine nitrile cathepsin K inhibitor and its application in terms of osteoarthritis is treated, belong to cathepsin inhibitors technical field.The inhibitor has new, different orientation P3 groups, in its extracellular depression effect to cathepsin K all in nanomolar concentrations, to K and S, B selectivity all hundreds times even more than.Even if the selectivity to homology very high cathepsin K and L also all more than hundreds times.Using Primary chondrocyte as model, using two kinds of condition of culture：(1) passage, (2) ascorbic acid and β phosphoglycerol stimulate culture.Gelatin zymography and the test of quantitative fluorescence method is respectively adopted, find the expression quantity of its cathepsin K has increase under two conditions；Then add after the new inhibitor, the activity reduction of cathepsin K.Illustrate that the inhibitor can also be played a role well in the cell, and good inhibition is shown to cathepsin K.

Description

Hydrazine nitrile cathepsin K inhibitor and its prepare treatment osteoarthritis drugs in Application

Technical field

The invention belongs to cathepsin inhibitors technical field, and in particular to the new and effective hydrazine nitrile tissue egg of a class White enzyme inhibitor, its selectivity to cathepsin K and B, L, S is greatly enhanced, and is preparing treatment osteoarthritis There is potential application in terms of related drugs, so as to clinically have important meaning for treatment osteoarthritis and relevant disease Justice.

Background technology

Osteoarthritis (Osteoarthritis, OA) is one caused by mechanical injuries and biological factor interaction Wide array of pathologies.As the disease that a kind of degenerated joint is disorderly, the principal character of osteoarthritis shows as articular cartilage damage, soft Bone sending down the fishbone change and synovitis etc., drastically influence the daily life of patient^[1]；Within the coming years, bone and joint diseases will The concern of people can be caused all the more^[2].OA pathogenesis is extremely complex, does not unify final conclusion also so far^[3].Treat OA's at present Medicine includes NSAIDs, glucocorticoid and some other bio-pharmaceutical (such as Glucosamine, chondroitin sulfate With hyaluronic acid etc.), but they are also only limitted to presentation treatment, and therapeutic effect is not obvious, poor reproducibility^[4]。

Evidence suggests cause the catabolism of cartilage at osteoarthritic inflammation due to the overexpression of proteolytic enzyme Ability strengthens, and includes the loss and the degraded of II collagen types of proteoglycans^[3,5].What is be identified at present causes cartilage degradation Key enzyme have：Matrix metalloproteinase (especially MMP-3, MMP-9 and MMP-13), proteoglycans enzyme (ADAMTS-4 and ) and cysteine proteinase ADAMTS-5^[5-8].Wherein a member --- the cathepsin of pawpaw class cysteine proteinase family K (Cathepsin K, Cat K), it is largely present in Various Tissues, such as：Bone, ovary, heart, placenta, lung cancer, skeletal muscle, Colon and small intestine^[9].When Cat K lack, pycnodysostosis can be caused^[10]With hardening disease^[11]；On the contrary, working as Cat During K over-expressions, the bone diseases such as osteoporosis, rheumatoid arthritis, atherosclerosis and osteoarthritis can be caused^[9,12], Therefore the normal expression of cathepsin K is most important in vital movement.There are some researches show Cat K can be in multiple positions The triple-helix structure of point cracking collagen^[13], thus as the drug targets enzyme for the treatment of osteoarthritis class disease；And it presses down Preparation then shows the ability for capitally protecting cartilage in terms of osteoarthritis is treated^[14-16], so that in bone biology research It is significant.

In order to provide the structure of more potential drug small molecules for treating osteoarthritis, press down also for its is further optimized Effect processed and biology performance, the present invention is with commercialized cathepsin K inhibitor --- and Odanacatib (ODNA) is ginseng According to the new small molecule inhibitors of the several new synthesis of comparative studies.

The content of the invention

The purpose of the present invention is：The first, two new cathepsin K inhibitors are provided --- (±) CKI-E and (±) CKI-F, they not only show good rejection (relatively low IC to cathepsin K₅₀And K_iValue), and to histone Enzyme K and B, L and S selectivity are greatly improved.2nd, the result of MTT colorimetric methods test shows, under conditions of high concentration presence Two kinds of inhibitor do not show any toxicity on a cellular level.3rd, gelatin zymography is utilized in mouse primary cartilage cell Activity influence of the two kinds of inhibitor tested with quantitative fluorescence method to cathepsin K.As a result show：Both inhibitor are all It is good medicine submerged body.

The synthesis of inhibitor of the present invention and sign

We change into the P3 positions group in novel inhibitors structure the methyl sulfone of contraposition/meta biphenyl, make inhibitor Bulk of molecule, orientation, hydrophilicity and hydrophobicity are more matched with the S3 pockets of cathepsin K in structure, so as to improve it to group Knit Proteinase K depression effect and they to cathepsin K and other cathepsins suppression selectivity.

The structural formula of the high selectivity novel inhibitors of heretofore described cathepsin K is as follows：

Specifically, including the following two kinds structure：

They can be obtained by following synthetic route：

Above route is to be directed to contraposition (para-) derivative and meta that synthesis P3 is (±) 4- methyl sulfone benzene respectively (meta-) derivative.In the building-up process of inhibitor small molecule, successively employ contraposition/meta bromo-acid and LDA is participated in Necleophilic reaction, the amidation process that participates in of isobutyl chlorocarbonate and N-methylmorpholine and aryl boric acid and aryl bromide ginseng With Suzuki react.

The inhibitor has new, different orientation P3 groups, in its extracellular depression effect to cathepsin K All in nanomolar concentrations, to K and S, B selectivity all hundreds times even more than.Even if to homology very high tissue Proteinase K and L selectivity are also all more than hundreds times.When the new inhibitor concentration is up to 10^-6During M, it is thin to osteogenic sarcoma Born of the same parents, the smooth muscle cell of ripe mouse or cartilage cell are without any side effects.Using Primary chondrocyte as model, use Two kinds of condition of culture：(1) passage, (2) ascorbic acid and β-phosphoglycerol stimulate culture.Be respectively adopted gelatin zymography and Quantitative fluorescence method is tested, and find the expression quantity of its cathepsin K has increase under two conditions；Then the new suppression is added After preparation, the activity reduction of cathepsin K.Illustrate that the inhibitor can also play a role well in the cell, to tissue egg White enzyme K shows good inhibition.

Brief description of the drawings

Fig. 1：Compound CKI-E inhibiting cathepsins K half-inhibition concentration (IC₅₀) experiment curv.

Fig. 2：Compound CKI-E inhibiting cathepsins L half-inhibition concentration (IC₅₀) experiment curv.

Fig. 3：Compound CKI-E inhibiting cathepsins S half-inhibition concentration (IC₅₀) experiment curv.

Fig. 4：Compound CKI-E inhibiting cathepsins B half-inhibition concentration (IC₅₀) experiment curv.

Fig. 5：Compound CKI-F inhibiting cathepsins K half-inhibition concentration (IC₅₀) experiment curv.

Fig. 6：Compound CKI-F inhibiting cathepsins L half-inhibition concentration (IC₅₀) experiment curv.

Fig. 7：Compound CKI-F inhibiting cathepsins S half-inhibition concentration (IC₅₀) experiment curv.

Fig. 8：Compound CKI-F inhibiting cathepsins B half-inhibition concentration (IC₅₀) experiment curv.

Fig. 9：Inhibitor (±) CKI-E and (±) CKI-F to the smooth muscle cell of ripe mouse, OS-732 cells, The cell survival rate test chart of mouse primary cartilage cell.

Figure 10：Cartilage cell in succeeding generations (primary P0, first generation P1, second generation P2, third generation P3) variation diagram.

Figure 11 gelatin zymographies test control group DMSO (A), compound CKI-E (B), CKI-F (C), ODAN (D) are to cartilage The active influence figure of cathepsin K in cell (P0, P1, P2, P3).

The activity expression figure of cathepsin K in Figure 12 (A) fluorescence method test cartilage cell (P0, P1, P2, P3)；(B) with DMSO is control group, fluorescence spectrometry compound CKI-E, CKI-F, and ODAN is to Cathepsin K activities in 2nd generation cartilage cell Expression figure.

The Primary chondrocyte and control group that the test of Figure 13 (A) fluorescence method was stimulated through ascorbic acid and β-phosphoglycerol are not The activity expression figure of cathepsin K in the Primary chondrocyte being stimulated；(B) using the Primary chondrocyte that stimulated as pair According to group, compound CKI-E, CKI-F is separately added into, after ODAN is suppressed, the activity of cathepsin K in each group cartilage cell Expression figure.

The depression effect detection of inhibitor

1. depression effect

Inhibitor mainly passes through IC to the depression effect of enzyme₅₀And K_iValue represents, wherein IC₅₀It is half-inhibition concentration, refers to The concentration of required inhibitor when the activity of enzyme being dropped into original vigor half under certain experiment condition；K_iIt is that inhibitor is made with enzyme The inhibition constant of dissociation constant, also referred to as inhibitor.

2. external Enzyme activity assay condition

Cathepsin K：Prepare 40mL, pH value for 5.5 MES-NaOH cushioning liquid it is stand-by, include 2.5mM EDTA, 2.5mM DTT and 10%DMSO；Using 20 μm of ol/L Z-Phe-Arg-AMC as fluorogenic substrate (note：All cushioning liquid of this patent In percentage used be all percent by volume).

Cathepsin L：Testing conditions, fluorogenic substrate are identical with cathepsin K.

Cathepsin B：Prepare 40mL, pH value for 6.0 MES-NaOH cushioning liquid it is stand-by, include 2.5mM EDTA, 2.5mM DTT, 10%DMSO and 0.001%Tween20；Using 20 μm of ol/L Z-Phe-Arg-AMC as fluorogenic substrate.

Cathepsin S：Prepare 40mL, pH value for 6.5 MES-NaOH cushioning liquid it is stand-by, include 2.5mM EDTA, 2.5mM DTT, 10%DMSO and 0.001%BSA；Using 40 μm of ol/L Z-VVR-AMC as fluorogenic substrate.

3. depression effect detection process

The stock solution of enzyme, substrate and inhibitor is configured first；Then as needed by inhibitor by a series of concentration ladder Degree dilution, the reaction rate of enzyme is monitored using ELIASA；Mapped according to the residual activity of enzyme and inhibitor concentration, calculate enzyme Half-inhibition concentration IC₅₀, and then calculate K_i；And structure-effect relationship analysis is carried out to it, final choice goes out depression effect height and choosing The good inhibitor of selecting property.

Set after instrument parameter, carry out Enzyme activity assay：

First, it is parallel in first three hole of 96 orifice plates to do 3 blank control group experiments, add successively in 9 holes below Enter the inhibitor (CKI-E or CKI-F) of 1 μ L various concentrations (1nM~7 μM)；Again the same of 10 μ L is separately added into this 12 holes A kind of enzyme (cathepsin K, L, B or S)；Then sequentially add the corresponding cushioning liquid of 40 μ L, rear 9 in preceding 3 holes respectively The corresponding cushioning liquid of 39 μ L is sequentially added in individual hole, makes the cumulative volume of solution in each hole up to 50 μ L.96 orifice plates are put into It is incubated in 37 DEG C of constant incubators after 30min, adds the corresponding substrates of 50 μ L；After fully mixing, using ELIASA immediately Carry out Enzyme assay.Carry out curve fitting, the data input Origin softwares measured in acquisition using Sigmoidal On curve, the concentration of required inhibitor, i.e. IC when observation enzyme activity is suppressed 50%₅₀Value.Inhibitor (CKI-E or CKI-F) is right Half-inhibition concentration (the IC of cathepsin K₅₀) experiment curv is shown in accompanying drawing 1-8.

4. Enzyme assay result is as shown in table 1：

Table 1：IC of the inhibitor to four kinds of enzymes₅₀Data

According to Michaelis constant (K having reported, measuring under the same conditions_m)^[17]：Cathepsin K, K_m=18.06 ± 0.22μM；Cathepsin L, K_m=3.525 ± 0.405 μM；Cathepsin B, K_m=157.5 ± 2.5 μM；Cathepsin S, K_m=102.19 ± 1.51 μM；And formula K_i=IC₅₀/(1+[S]/K_m), by IC₅₀Value is converted into K_iThe results are shown in Table after value 2；Wherein [S] is concentration of substrate, K_mFor Michaelis constant.

Table 2：K of the inhibitor to four kinds of enzymes_iData

Known by result of the test, two new inhibitors synthesized by the present invention all reach to the inhibition of cathepsin K Nanomolar concentration rank is arrived, wherein (±) CKI-E inhibition constant has reached 1.14nM.And (±) CKI-E and (±) CKI-F is also fine for homology restriction endonuclease K and L selectivity, has respectively reached 195 times and 406 times.Two inhibitor are to group Proteinase K and S, B selectivity are knitted also all at hundreds times or higher, it is especially desirable to, it is noted that (±) CKI-F is to histone Enzyme K and B selectivity have reached 3345 times.

The measure of cell survival rate

1.MTT colorimetric methods

The present invention is using tetrazolium salts (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium Bromid, MTT) based on colorimetric method (Colorimetric assay) detect toxicity of the inhibitor small molecule to cell； Its principle is：In the living cells with metabolic activity, the MTT of yellow can be cleaved empurpled first a ceremonial jade-ladle, used in libation (Formazan) crystalline substance Body^[18,19]；This crystal, which is soluble in DMSO, forms colored solutions, is then quantitatively detected with porous scanning spectrophotometer.

2. cell model and condition of culture

The present invention uses three kinds of cell models, is respectively：(a) smooth muscle cell (the Mouse smooth of ripe mouse Muscle cell line MOVAS, are obtained from the smooth muscle of ripe mouse), (b) OS-732 cells (Human Osteosarcoma MG-63cell line, are obtained from human osteblast's sarcoma), (c) mouse primary cartilage cell (Primary Chondrocytes, is obtained at the cartilage of the young mouse of firm birth 5-6 days).

The condition of culture of cell：These three cell (smooth muscle cell, osteosarcoma cells are cultivated in 12 porocyte culture plates Or Primary chondrocyte), a class cell is only cultivated per plate, and use same high glycoform DMEM culture mediums (10% hyclone； 100U/mL penicillin and 100 μ g/mL streptomysins；1% glutamine；2%HEPES buffer solutions), wherein 50000 thin per hole Born of the same parents, 1mL culture mediums；Culture environment is to contain 5%CO₂With 37 DEG C of constant incubators of 95% air.One is changed after 48 hours Subculture (note：Percentage used is all percent by volume in all culture mediums of this patent and culture cell condition).

3.MTT colorometric assay processes

When cell quantity grows to about 100 000/hole, the test of cell survival rate is carried out using MTT colorimetric methods.First Two control groups (with or without DMSO) are set, to observe whether DMSO has an impact to the activity of cell；Secondly by present invention system Standby micromolecular inhibitor (±) CKI-E and (±) CKI-F DMSO dissolves, and is configured to 10^-3Mol/L storing solutions are stand-by；Then DMSO and inhibitor C KI-E, CKI-F storing solution is added into DMEM culture mediums, each row are containing 3 holes as parallel, and totally 4 row are allowed to Concentration gradient is presented：10^-9、10^-8、10^-7、10^-6mol/L；After cell quantity and state up to standard, upper strata culture medium is removed, 12 1mL gradients containing various concentrations DMSO and inhibitor C KI-E, CKI-F cell training are separately added into every hole of porocyte culture plates Support base.Each concentration has three Duplicate Samples, so testing 3 times (n=9) altogether；Incubator is put into afterwards is incubated 48h.Added per hole 25 μ L MTT (5mg/mL, tetrazolium salts, yellow), continue to be incubated 4h.Culture medium is removed, there is water insoluble purple first a ceremonial jade-ladle, used in libation crystal； Then 1mL DMSO are added per hole and dissolves this crystal, the depth of color is directly proportional to the content of first a ceremonial jade-ladle, used in libation.Until completely dissolved, next day The use of ELIASA in wavelength is that the light absorption value of sample is measured at 570nm.In the range of certain cell quantity, sample Light absorption value is directly proportional to cell survival rate.

4. experimental result is with discussing

Smooth muscle cells, OS-732 cells, mouse of inhibitor (±) CKI-E and (±) CKI-F to ripe mouse The test result of the cell survival rate of Primary chondrocyte is shown in accompanying drawing 9.As a result show：When concentration is 10^-9~10^-6Mol/L scopes Interior, DMSO does not produce interference to MTT colorimetric methods.Then the cell survival rate of the control group containing DMSO is set as 100%, plus Enter the ratio as cell survival rate of the absorption value of the experimental group of inhibitor and the absorption value of unchecked dose of control group.When dense Two kinds of micromolecular inhibitors all do not produce obvious shadow to the cell survival rate of three kinds of cells when degree is gradually increased to 1000nM by 1nM Ring, this shows in this concentration range, and two kinds of inhibitor are non-toxic to the three kinds of cells tested.

The active testing of intracellular cathepsin K

Using mouse primary cartilage cell as model, the present invention is using gelatin zymography and quantitative fluorescence method test inhibitor The active influence of CKI-E and CKI-F on a cellular level to cathepsin K.

1. method of testing-gelatin zymography and quantitative fluorescence method brief introduction

Gelatin zymography^[20]It is to be based on mixing protein substrate (gelatin) with SDS- polyacrylamide gels, makes to be measured The method of protein sample electrophoresis in this gel.Protein is combined generation protein-SDS with anion-containing SDS in gel Intermediate product, causes and a large amount of negative electrical charges is carried on testing protein so that the quantity of electric charge considerably beyond protein in itself, so that The protein migration speed in running gel is set to be only dependent upon the molecular weight of itself, so as to ignore protein in itself because of electric charge Different and difference in the migration rate brought.After gel electrophoresis, with eluent remove SDS, testing protein it is active extensive It is multiple.It is incubated after a period of time, testing protein is fully hydrolyzed substrate gelatin, then with coomassie brilliant blue staining, you can display one Bar clearly white ribbon.According to the density and area of this white ribbon, the activity of enzyme just can be determined^[21]。

Quantitative fluorescence method is a kind of new method, can be with the work of selective enumeration method Primary chondrocyte inner tissue Proteinase K Property.Its general principle is as follows：Under conditions of 5- nitrosalicylaldehydes (5-Nitrosalicylaldehyde, abbreviation NSA) are present, By cell and substrate-peptide derivant (4-methoxy- β-naphthylamine, abbreviation 4M β NA) of test serum Proteinase K It is common to be incubated the light absorption value for determining its fluorescence-causing substance after a period of time with XRF, it is used as control experiment value.Then add After the micromolecular inhibitor of cathepsin K, the absorption value of re-test fluorescence-causing substance.Finally, by adding inhibitor and not adding Absorption value ratio calculation (the Enzyme activities of inhibitor:Obtain group Knit the activity of protease.

2. the condition of culture of cell

The culture of Primary chondrocyte (includes 4.5g/L glucose, 10% hyclone, 1% green grass or young crops using DMEM culture mediums Mycin-streptomysin, 1% glutamate, 1%HEPES buffer solutions), continuous culture 10 days, as a control group system；Then use Same DMEM culture mediums, but contain 50 μ g/mL ascorbic acid (Ascorbic acid, AA) and 7.5mM β-phosphoglycerol simultaneously (β-glycerophosphate, β-GP), continuous culture 10 days, as experimental group system, act as stimulating inducing cell to produce More cathepsins.At the same time, the present invention is also passed on to cartilage cell, and (4.5g/ is included using DMEM culture mediums L Glucose, 10% hyclone, 100U/mL penicillin and 100 μ g/mL streptomysins, 1% glutamate, 1%HEPES bufferings Liquid；Passage step all uses this culture medium below) culture about 48h.After remove this culture medium, after being cleaned with PBS, add 2mL pancreatin (Trypsin) being placed in after in 37 DEG C of incubators waits cell to suspend, and then adds the piping and druming of 8mL DMEM in high glucose culture medium and mixes. Divide 5mL mixed liquors to centrifuge (400g, 5min) in 15mL centrifuge tubes, obtain lower sediment, and add 500 μ L Extraction buffers (20nM Tris-HCl, pH=7.5,5mM EGTA, 150mM NaCl, 10mM NaF, 20mM β-glycerol phosphate, 1mM sodium orthovanadate, 1%Triton X-100,0.1%Tween 20), save as P0 (Passage 0) with Make standby.Remaining 5mL mixed liquors are placed in new culture dish, 4~5mL DMEM in high glucose culture mediums is added and is placed in incubator In (be labeled as Passage 1, P1), for passing on.It is (attached：P1 preservation, and P2, P3 preservation are passed on above-mentioned step Suddenly).Cartilage cell Primary chondrocytes in succeeding generations (the primary, first generation, the second generation, the third generation) change See accompanying drawing 10.

3. result is with discussing

3.1 using by the cartilage cell (primary P0, first generation P1, second generation P2, third generation P3) before and after passage, using bright Glue zymography tests the activity of cathepsin K therein respectively.Control group (A) using DMSO as without inhibitor, experiment Group is separately added into 1 μM of inhibitor (B:ODAN、C:CKI-E、D:CKI-F), experimental result is shown in accompanying drawing 11.

Understood from figure (A), by primary P0 to second generation P2 for cartilage cell inner tissue Proteinase K it is active with algebraically gradually Enhancing, and the expression quantity highest in the second generation.When adding inhibitor ODAN (B), CKI-E (C), CKI-F (D) and concentration reaches 1 μ During M, the activity of cathepsin is substantially suppressed.

3.2 uses are by the cartilage cell (primary P0, first generation P1, second generation P2, third generation P3) before and after passage, using calmly Measure the activity that fluorescence method tests cathepsin K therein.Using DMSO as the control group of without inhibitor, and it is separately added into 1 μM Sample with 2 μM of inhibitor (ODAN, CKI-E, CKI-F) is as experimental group, and test result is shown in accompanying drawing 12.

It was found from figure (A) result：Activity from primary P0 to second generation P2 for cartilage cell inner tissue Proteinase K gradually increases By force, the expression quantity highest and in the second generation.Then when being separately added into 1 μM, after 2 μM of inhibitor (ODAN, CKI-E, CKI-F), it The depression effect of intracellular cathepsin K is strengthened with the rise of inhibitor concentration, see figure (B).

3.3 using 50 μ g/mL ascorbic acid (Ascorbic acid, AA) and 7.5mM β-phosphoglycerol (β- Glycerophosphate, β-GP) continued stimulus induce 10 days cartilage cell and without stimulate induction cartilage cell's conduct Experimental comparison group, the expression quantity of intracellular cathepsin K is detected using quantitative fluorescence method, and test result is shown in accompanying drawing 13.

As a result (A) shows：When using without stimulating the cell of induction as check experiment group, through the cell for stimulating induction The relative activity of interior cathepsin K is substantially increased by 100 expression quantity for increasing to 140, i.e. cathepsin K.Then, respectively 0.1 μM or 1 μM of inhibitor (CKI-E, CKI-F, ODAN) is sequentially added, their suppression to intracellular cathepsin K Effect strengthens with the rise of inhibitor concentration, sees figure (B).

3.4 in summary gelatin zymography and quantitative fluorescence method test result indicates that：In the cartilage cell passed on, The expression quantity of cathepsin K is in P2 up to maximum；When adding after inhibitor ODAN, CKI-E or CKI-F, they can suppress The activity of intracellular cathepsin K.And after cartilage cell is stimulated by induction, the expression of intracellular cathepsin K Amount is significantly increased；Equally, when adding after ODAN, CKI-E or CKI-F, they also can inhibiting cathepsin K under the conditions of being somebody's turn to do Activity.

Embodiment

On the premise of existing (±) E4 (para-/meta-) synthesizes^[26], take 0.61g parent compounds (±) E4 And other compound obtained from commercial channel (para-)：0.43g is to (±) methyl sulfone phenyl boric acid ((4- (methylsulfonyl) phenyl) boronic acid), 65.3mg [1,1'- double (diphenylphosphine) ferrocene] palladium chloride (Pd(dppf)Cl₂) and 0.5g potassium carbonate powders be separately added into stirring magneton 100mL round-bottomed flasks in, then by 40mL Tetrahydrofuran add round-bottomed flask in as reaction dissolvent, then with liquid-transfering gun inject 2mL water.Three times (nitrogen-vacuum) fills and taken out After reaction system, about 6h is heated to reflux^[27].And reaction process is monitored with thin-layer chromatography (TLC), question response is complete, with rotation Tetrahydrofuran in flask is evaporated by evaporimeter.Then 60mL ethyl acetate and 20mL water are added, is separated with separatory funnel organic Phase, then 3 aqueous phases are extracted with ethyl acetate, merge organic phase.And washed successively with water, saturated sodium bicarbonate and saturated sodium-chloride Organic phase.It is to be dried to finish with anhydrous sodium sulfate drying, this drier is filtered out, ethyl acetate solvent is evaporated, compound is obtained (±) CKI-E crude product；Finally use petroleum ether：Ethyl acetate=1：1 (volume：Volume) mixed solvent as solvent, Silica gel column chromatography purification is carried out, (±) CKI-E white powdery solids 0.23g is obtained.

Same step, (±) E4 (para-) is replaced with (±) E4 (meta-), and other experiment conditions are identical, obtain (±) CKI-F white powdery solids 0.19g.

CKI-E characterization results are as follows：

¹H NMR(500MHz,CDCl₃):δ 8.03 (d, J=8.2Hz, 2H), δ 7.78 (d, J=8.3Hz, 2H), δ 7.61 (d, J=8.2Hz, 2H), δ 7.46 (dd, J=22.0,9.8Hz, 2H), δ 4.26 (t, J=7.2Hz, 1H), δ 3.21 (s, 4H), δ 3.12 (s, 3H), δ 2.62 (s, 3H), δ 2.09-1.98 (m, 1H), δ 1.66 (dt, J=29.6,11.4Hz, 1H), δ 1.52 (dt, J=13.7,7.0Hz, 1H), δ 1.01-0.94 (m, 6H)

¹³C NMR(126MHz,CDCl₃):δ174.48(s),δ145.85(s),δ140.32(s),δ139.28(s),δ 137.99 (s), δ 128.74 (s), δ 127.91 (d, J=18.6Hz), δ 113.91 (s), δ 45.81 (s), δ 44.61 (s), δ 43.58(s),δ40.59(s),δ30.64(s),δ25.67(s),δ22.71(s),δ22.40(s).

MS(ESI)m/z:414.1841[M+H]⁺,827.3567[2M+H]⁺.

CKI-F characterization results are as follows：

¹H NMR(500MHz,CDCl₃):δ 8.04 (d, J=8.1Hz, 2H), δ 7.80 (dd, J=18.9,8.1Hz, 1H), δ 7.58-7.51 (m, 2H), δ 7.47 (dd, J=15.1,7.4Hz, 1H), δ 4.30 (dt, J=14.8,7.2Hz, 1H), δ 3.20 (s, 3H), δ 3.12 (s, 4H), δ 2.59 (s, 2H), δ 2.15-1.81 (m, 2H), δ 1.50 (ddd, J=22.6,14.0,7.2Hz, 1H), δ 0.97 (dd, J=10.9,4.3Hz, 6H)

¹³C NMR(126MHz,CDCl₃):δ174.50(s),δ146.02(s),δ140.75(s),δ139.87(s),δ 139.49 (s), δ 129.69 (s), δ 128.54-127.67 (m), 126.61 (d, J=8.7Hz), δ 126.29 (s), δ 46.19 (s),δ44.64(s),δ43.73(s),δ40.57(s),δ30.67(s),δ25.74(s),δ22.64(s),δ22.47(s).

MS(ESI)m/z:414.1839[M+H]⁺,827.3571[2M+H]⁺.

Bibliography

1.Johnson,V.L.；Hunter,D.J.；Best Pract.Res.Cl.Rh.,2014,28,5.

2.Mueller,M.B.；Tuan,R.S.；Pm.R.,2011,3,S3.

3.Ishiguro,N.；Kojima,T.；Poole,A.R.；Nagoya J.Med.Sci.,2002,65,73.

4.Martel-Pelletier,J.；Lajeunesse,D.；Fahmi,H.；Tardif,G.；Pelletier, J.P.；Curr.Rheumatol.Rep.,2006,8,30.5.

5.Loeser,R.F.；J.Musculoskel.Neuron.,2008,8,303.6.

6.Miller,R.E.；Lu,Y.；Tortorella,M.D.；Malfait,A.M.；Curr.Rheumatol.Rep., 2013,15,350.

7.Zhou,S.；Thornhill,T.S.；Meng,F.；Xie,L.；Wright,J.；Glowacki,J.； J.Orthop.Res.,2016,34,454.

8.Kozawa,E.；Cheng,X.W.；Urakawa,H.；Arai,E.；Yamada,Y.；Kitamura,S.；Sato, K.；Kuzuya,M.；Ishiguro,N.；Nishida,Y.；J.Orthop.Res.,2016,34,127.

9.Yasuda,Y.；Kaleta,J.；D.；Adv.Drug Deliver.Rev.,2005,57,973.

10.Gelb,B.D.；Shi,G.P.；Chapman,H.A.；Desnick,R.J.；Science,1996,273, 1236.

11.Gowen,M.；Lazner,F.；Dodds,R.；Kapadia,R.；Field,J.；Tavaria,M.； Bertoncello,I.；Drake,F.；Zavarselk,S.；Tellis,I.；Hertzog,P.；Debouck,C.；Kola, I.；J.Bone Miner.Res.,1999,14,1654.

12.Vasiljeva,O.；Reinheckel,T.；Peters,C.；Turk,D.；Turk,V.；Turk,B.；Curr Pharm Des.,2007,13,387.

13.Novinec,M.；B.；Biol.Chem.,2013,394,1163.

14.Hayami,T.；Zhuo,Y.；Wesolowski,G.A.；Pickarski,M.；Duong T.；Bone,2012, 50,1250.

15.McDougall,J.J.；Schuelert,N.；Bowyer,J.；Osteoarthr.Cartilage,2010, 18,1355.

16.Connor,J.R.；LePage,C.；Swift,B.A.；Yamashita,D.；Bendele,A.M.；Maul, D.；Kumar,S.；Osteoarthr.Cartilage,2009,17,1236.

17.Ren X.-F.；Li H.-W.；Fang X.；Wu Y.；Wang L.；Zou S.；Org.Biomol.Chem., 2013,11,1143.

18.Vistica,D.T.；Skehan,P.；Scudiero,D.；Monks,A.；Pittman,A.；Boyd,M.R.； Cancer Res.,1991,51,2515.

19.Slater,T.F.；Sawyer,B.；U.；Biochim.Biophys.Acta,1963,77,383.

20.Kleiner,D.E.；Stetler-stevenson,W.G.；Anal.Biochem.,1994,218,325.

21. Chen Qiu phoenix；Week is shockproof；Hubei Institute For Nationalities's journal, 2012,29,70.

22.Rüttger,A.；Mollenhauer,J.；R.；Gütschow,M.；Wiederanders,B.； Biotechniques,2006,41,469.

23.Dolbeare,F.A.；Smith,R.E.；Clin.Chem.,1977,23,1485.

24.Schwartz,W.N.；Barrett,A.J.；Biochem.J.,1980,191,487.

25.Simon,J.；Duffy,M.J.；Biochem.Soc.Trans.,1986,14,460.

26.Yuan,X.-Y.；Fu,D.-Y.；Ren X.-F.；Fang X.；Wang L.；Zou S.；Wu Y.； Org.Biomol.Chem.,2013,11,5847.

27.Kaupp,G.；Naimi-Jamal,M.R.；Stepanenko,V.；Chem.Eur.J.,2003,9,1156.

Claims (3)

1. a kind of hydrazine nitrile cathepsin inhibitors, its structural formula is as follows：

2. a kind of hydrazine nitrile cathepsin inhibitors as claimed in claim 1, its structural formula is as follows：

3. application of the hydrazine nitrile cathepsin inhibitors in treatment osteoarthritis drugs are prepared described in claim 1 or 2.