CN105837479A - Hydrazinonitrile cathepsin K inhibitor, and application thereof in preparation of osteoarthritis treatment drugs - Google Patents
Hydrazinonitrile cathepsin K inhibitor, and application thereof in preparation of osteoarthritis treatment drugs Download PDFInfo
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Abstract
The invention relates to a hydrazinonitrile cathepsin K inhibitor, and an application thereof in preparation of osteoarthritis treatment drugs, and belongs to the technical field of cathepsin inhibitors. The inhibitor contains a novel P3 group with different orientations, has a nano-mole concentration level inhibition effect on cathepsin K outside cells, has hundreds-of-times or above selectivity to cathepsin K, cathepsin S and cathepsin B, and also has hundreds-of-times or above selectivity to highly homologous cathepsin K and cathepsin L. Culture is carried out under the following two conditions: (1) cell passage and (2) ascorbic acid and beta-glycerophosphoric acid stimulation with primary chondrocytes as a model. Results of gelatin zymography and quantitative fluorescence assay tests show that the expression level of the cathepsin K under every one of the above two conditions is increased; and the activity of the cathepsin K is reduced after the novel inhibitor is added. The inhibitor also has a good inhibition effect in the cells, and has a very good inhibition effect on the cathepsin K.
Description
Technical field
The invention belongs to cathepsin inhibitors technical field, it is specifically related to the hydrazine nitrile cathepsin inhibitors that a class is new and effective, the selectivity of cathepsin K and B, L, S is greatly enhanced by it, and in terms of the related drugs of preparation treatment osteoarthritis, have potential application, thus there is important meaning for treatment osteoarthritis and relevant disease clinically.
Background technology
Osteoarthritis (Osteoarthritis, OA) is a series of pathological changes being interacted by mechanical injuries and biological factor and being caused.As the disease that a kind of degenerated joint is disorderly, the principal character of osteoarthritis shows as articular cartilage damage, subchondral bone change and synovitis etc., drastically influence the daily life of patient[1]；Within the coming years, joint disease will cause all the more the concern of people[2].The pathogenesis of OA is extremely complex, the most ununified final conclusion[3].The medicine for the treatment of OA includes NSAID (non-steroidal anti-inflammatory drug), glucocorticoid and some other bio-pharmaceutical (such as glucosamine, chondroitin sulfate and hyaluronic acid etc.) at present, but they are also only limitted to presentation treatment, and therapeutic effect is inconspicuous, poor reproducibility[4]。
Evidence suggests, cause due to the overexpression of proteolytic enzyme the catabolism ability of cartilage to strengthen at osteoarthritic inflammation, including loss and the degraded of II collagen type of Dan Baiduotang proteoglycan PG[3,5].The key enzyme causing cartilage degradation being identified at present has: matrix metalloproteinase (especially MMP-3, MMP-9 and MMP-13), Dan Baiduotang proteoglycan PG enzyme (ADAMTS-4 and ADAMTS-5) and cysteine proteinase[5-8].Wherein a member cathepsin K (Cathepsin K, Cat K) of Fructus Chaenomelis class cysteine proteinase family, it is present in Various Tissues in a large number, such as: bone, ovary, heart, Placenta Hominis, pulmonary carcinoma, skeletal muscle, colon and small intestinal[9].When Cat K lacks, pycnodysostosis can be caused[10]With hardening disease[11]；On the contrary, when Cat K over-expression, the osteopathias 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 multiple sites cracking collagen protein triple-helix structure[13], thus become the drug targets enzyme for the treatment of osteoarthritis class disease；Its inhibitor then shows the ability protecting cartilage capitally in terms for the treatment of osteoarthritis[14-16], thus significant in bone biology is studied.
In order to provide the structure of the little molecule of potential drug of more treatment osteoarthritis, also for optimizing its depression effect and biology performance further, the present invention with business-like cathepsin K inhibitor Odanacatib (ODNA) as reference, the several newly synthesized new small molecule inhibitors of comparative study.
Summary of the invention
It is an object of the invention to: one, provide two novel cathepsin K inhibitors (±) CKI-E and (±) CKI-F, they not only show good rejection (relatively low IC to cathepsin K50And KiValue), and the selectivity of cathepsin K and B, L and S is greatly improved.Two, the result of MTT colorimetry test shows, under conditions of high concentration exists, two kinds of inhibitor the most all do not show any toxicity.Three, in mouse primary chondrocyte, utilize two kinds of inhibitor that gelatin zymography and quantitative fluorescence method tested activity influence to cathepsin K.Result shows: both inhibitor are all good medicine submerged bodies.
The synthesis of inhibitor of the present invention and sign
P3 position group in novel inhibitors structure is changed into the methyl sulfone of para-position/meta biphenyl by us, make the size of inhibitor molecules, orientation, hydrophilicity and hydrophobicity the most more mate with the S3 pocket of cathepsin K, thus improve it to the depression effect of cathepsin K and their suppression selectivitys to cathepsin K and other cathepsin.
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 be respectively for synthesis P3 position be (±) para-position (para-) derivant of 4-methyl sulfone benzene and meta (meta-) derivant.In the building-up process of the little molecule of inhibitor, successively have employed para-position/meta bromo-acid and the Suzuki reaction of the necleophilic reaction of LDA participation, isobutyl chlorocarbonate and the amidation process of N-methylmorpholine participation and aryl boric acid and aryl bromide participation.
This inhibitor has P3 group novel, different orientation, in extracellular its to the depression effect of cathepsin K all nanomolar concentrations, to the selectivity of K and S, B all hundreds times even more than.Even if the selectivity of the cathepsin K the highest to homology and L is the most all more than hundreds times.When this new inhibitor concentration reaches 10-6During M, it is the most without any side effects to human osteosarcoma cell, the smooth muscle cell of ripe mice or chondrocyte.With Primary chondrocyte as model, use two kinds of condition of culture: (1) passage, (2) ascorbic acid and β-phosphoglycerol stimulate to be cultivated.It is respectively adopted gelatin zymography and the test of quantitative fluorescence method, finds that the expression of its cathepsin K has increase under two conditions；After being subsequently adding this new inhibitor, the activity reduction of cathepsin K.Illustrate also can play a role well at this inhibitor intracellular, cathepsin K is shown good inhibition.
Accompanying drawing explanation
Fig. 1: the half-inhibition concentration (IC of compound CKI-E inhibiting cathepsin K50) experiment curv.
Fig. 2: the half-inhibition concentration (IC of compound CKI-E inhibiting cathepsin L50) experiment curv.
Fig. 3: the half-inhibition concentration (IC of compound CKI-E inhibiting cathepsin S50) experiment curv.
Fig. 4: the half-inhibition concentration (IC of compound CKI-E inhibiting cathepsin B50) experiment curv.
Fig. 5: the half-inhibition concentration (IC of compound CKI-F inhibiting cathepsin K50) experiment curv.
Fig. 6: the half-inhibition concentration (IC of compound CKI-F inhibiting cathepsin L50) experiment curv.
Fig. 7: the half-inhibition concentration (IC of compound CKI-F inhibiting cathepsin S50) experiment curv.
Fig. 8: the half-inhibition concentration (IC of compound CKI-F inhibiting cathepsin B50) experiment curv.
Fig. 9: inhibitor (±) CKI-E and (±) CKI-F is to the ripe smooth muscle cell of mice, OS-732 cells, the cell survival rate test figure of mouse primary chondrocyte.
Figure 10: chondrocyte is the variation diagram of (primary P0, first generation P1, second filial generation P2, third generation P3) in succeeding generations.
Figure 11 gelatin zymography test comparison group DMSO (A), compound CKI-E (B), CKI-F (C), ODAN (D) in chondrocyte (P0, P1, P2, P3) cathepsin K activity affect figure.
The activity expression figure of cathepsin K in Figure 12 (A) fluorescence method test chondrocyte (P0, P1, P2, P3)；(B) with DMSO as matched group, fluorescence spectrometry compound CKI-E, CKI-F, ODAN is to the expression figure of Cathepsin K activities in 2nd generation chondrocyte.
The activity expression figure of cathepsin K in Primary chondrocyte that the test of Figure 13 (A) fluorescence method stimulated through ascorbic acid and β-phosphoglycerol and the Primary chondrocyte that matched group was not stimulated；(B) with the Primary chondrocyte that stimulated as matched group, it is separately added into compound CKI-E, CKI-F, after ODAN suppresses, the activity expression figure of cathepsin K in each group chondrocyte.
The depression effect detection of inhibitor
1. depression effect
The depression effect of enzyme is mainly passed through IC by inhibitor50And KiValue represents, wherein IC50It is half-inhibition concentration, refers to the activity of enzyme be dropped to the concentration of required inhibitor during original vigor half under certain experiment condition；KiIt it is the inhibition constant of the dissociation constant of inhibitor and enzyme effect, also referred to as inhibitor.
The most external Enzyme activity assay condition
Cathepsin K: preparation 40mL, pH value be 5.5 MES-NaOH buffer solution stand-by, include 2.5mM EDTA, 2.5mM DTT and 10%DMSO；With the Z-Phe-Arg-AMC of 20 μm ol/L as fluorogenic substrate (note: in all buffer solution of this patent, percentage ratio used is all percent by volume).
Cathepsin L: testing conditions, fluorogenic substrate are all identical with cathepsin K.
Cathepsin B: preparation 40mL, pH value be 6.0 MES-NaOH buffer solution stand-by, include 2.5mM EDTA, 2.5mM DTT, 10%DMSO and 0.001%Tween20；With the Z-Phe-Arg-AMC of 20 μm ol/L as fluorogenic substrate.
Cathepsin S: preparation 40mL, pH value be 6.5 MES-NaOH buffer solution stand-by, include 2.5mM EDTA, 2.5mM DTT, 10%DMSO and 0.001%BSA；With the Z-VVR-AMC of 40 μm ol/L as fluorogenic substrate.
3. depression effect detection process
First the stock solution of enzyme, substrate and inhibitor is configured；The most as required inhibitor is diluted by a series of Concentraton gradient, use microplate reader to monitor the reaction rate of enzyme；Residual activity according to enzyme and inhibitor concentration mapping, calculate the half-inhibition concentration IC of enzyme50, and then calculate Ki；And it is carried out structure-effect relationship analysis, finally select the inhibitor that depression effect is high and selectivity is good.
After instrument parameter is set, carry out Enzyme activity assay:
First, parallel in first three hole of 96 orifice plates do the experiment of 3 blank groups, 9 holes are sequentially added into below the inhibitor (CKI-E or CKI-F) of 1 μ L variable concentrations (1nM～7 μMs)；The same enzyme (cathepsin K, L, B or S) of 10 μ L it is separately added into again in these 12 holes；It is sequentially added into 40 buffer solution corresponding for μ L in front 3 holes the most respectively, rear 9 holes are sequentially added into 39 buffer solution corresponding for μ L, make the cumulative volume of solution in each hole reach 50 μ L.96 orifice plates are put into after 37 DEG C of constant incubators hatch 30min, add 50 substrates corresponding for μ L；Fully after mixing, microplate reader is utilized to carry out Enzyme assay immediately.The data input Origin software that will record, utilizes Sigmoidal to carry out curve fitting, and on the curve obtained, observes enzyme activity and is suppressed the concentration of required inhibitor, i.e. IC when 50%50Value.The inhibitor (CKI-E or the CKI-F) half-inhibition concentration (IC to cathepsin K50) experiment curv is shown in accompanying drawing 1-8.
4. Enzyme assay result is as shown in table 1:
Table 1: the inhibitor IC to four kinds of enzymes50Data
According to Michaelis constant (K that reported, that record under the same conditionsm)[17]: cathepsin K, Km=18.06 ± 0.22 μM；Cathepsin L, Km=3.525 ± 0.405 μM；Cathepsin B, Km=157.5 ± 2.5 μMs；Cathepsin S, Km=102.19 ± 1.51 μMs；And formula Ki=IC50/(1+[S]/Km), by IC50Value is converted into KiThe results are shown in Table 2 after value；Wherein [S] is concentration of substrate, KmFor Michaelis constant.
Table 2: the inhibitor K to four kinds of enzymesiData
Being known by result of the test, two new inhibitors synthesized by the present invention have all reached nanomolar concentration rank to the inhibition of cathepsin K, wherein (±) inhibition constant of CKI-E reached 1.14nM.And (±) CKI-E and (±) CKI-F is also fine for the selectivity of homology restriction endonuclease K and L, has respectively reached 195 times and 406 times.Two inhibitor to the selectivity of cathepsin K and S, B the most all in hundreds times or higher, it is especially desirable to it is noted that (±) CKI-F reached 3345 times to the selectivity of cathepsin K and B.
The mensuration of cell survival rate
1.MTT colorimetry
The present invention uses tetrazolium salts (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid, MTT) based on colorimetry (Colorimetric assay) detect the inhibitor little molecule toxicity to cell；Its principle is: in the living cells with metabolic activity, and the MTT of yellow can cleaved empurpled first a ceremonial jade-ladle, used in libation (Formazan) crystal[18,19]；This crystal is soluble in DMSO being formed colored solutions, then by porous scanning spectrophotometer detection by quantitative.
2. cell model and condition of culture
The present invention uses three kinds of cell models, it is respectively as follows: smooth muscle cell (the Mouse smooth muscle cell line MOVAS of (a) ripe mice, obtain from the smooth muscle of ripe mice), (b) OS-732 cells (Human osteosarcoma MG-63cell line, obtain from human osteblast's sarcoma), (c) mouse primary chondrocyte (Primary chondrocytes obtains at the cartilage of the just birth young Mus of 5-6 days).
The condition of culture of cell: cultivating these three cell (smooth muscle cell, osteosarcoma cell or Primary chondrocyte) in 12 porocyte culture plates, every plate is only cultivated a class cell, and used same high glycoform DMEM culture medium (10% hyclone；100U/mL penicillin and 100 μ g/mL streptomycins；1% glutamine；2%HEPES buffer), wherein every 50000, hole cell, 1mL culture medium；Culture environment is containing 5%CO237 DEG C of constant incubators with 95% air.A subculture (note: in all culture medium of this patent and cultivation cell condition, percentage ratio used is all percent by volume) is changed after 48 hours.
3.MTT colorometric assay process
When cell quantity grows to about 100 000/hole, MTT colorimetry is utilized to carry out the test of cell survival rate.Two matched groups (with or without DMSO) are first set, whether the activity of cell are had an impact observing DMSO；Secondly micromolecular inhibitor of being prepared by the present invention (±) CKI-E and (±) CKI-F DMSO dissolves, and is configured to 10-3Mol/L storing solution is stand-by；Then the storing solution of DMSO and inhibitor C KI-E, CKI-F being added DMEM culture medium, every string is containing 3 holes as parallel, and totally 4 row are allowed to present Concentraton gradient: 10-9、10-8、10-7、10-6mol/L；When cell quantity and state up to standard after, remove upper strata culture medium, every hole of 12 porocyte culture plates be separately added into 1mL containing variable concentrations gradient DMSO and the cell culture medium of inhibitor C KI-E, CKI-F.Each concentration has three Duplicate Samples, so 3 times (n=9) of test altogether；Put into incubator afterwards and hatch 48h.Every hole adds 25 μ L MTT (5mg/mL, tetrazolium salts, yellow), continues to hatch 4h.Remove culture medium, have water-fast purple first a ceremonial jade-ladle, used in libation crystal；Then every hole addition 1mL DMSO dissolves this crystal, and the depth of color is directly proportional to the content of first a ceremonial jade-ladle, used in libation.Until completely dissolved, using microplate reader next day is that at 570nm, light absorption value to sample is measured at wavelength.In the range of certain cell quantity, the light absorption value of sample is directly proportional to cell survival rate.
4. experimental result and discussion
Inhibitor (±) CKI-E and (±) the ripe smooth muscle cell of mice, OS-732 cells, the test result of cell survival rate of mouse primary chondrocyte be shown in accompanying drawing 9 by CKI-F.Result shows: when concentration is 10-9～10-6In the range of mol/L, DMSO does not produces interference to MTT colorimetry.Then the cell survival rate of the matched group containing DMSO is set as 100%, and the absorption value of experimental group adding inhibitor is cell survival rate with the ratio of the absorption value of the matched group of unchecked dose.When concentration is gradually increased to 1000nM by 1nM, two kinds of micromolecular inhibitors the most not cell survival rate to three kinds of cells produces and significantly affects, and this shows in this concentration range, two kinds of inhibitor, three kinds of equal avirulences of cell to being tested.
The active testing of intracellular cathepsin K
With mouse primary chondrocyte as model, the present invention uses gelatin zymography and quantitative fluorescence method test inhibitor C KI-E and the CKI-F active impact on a cellular level on cathepsin K.
1. method of testing gelatin zymography and quantitative fluorescence method brief introduction
Gelatin zymography[20]It is to mix with SDS-polyacrylamide gel based on by protein substrate (gelatin), makes testing protein sample method of electrophoresis in this gel.Protein anion-containing SDS in gel is combined generation protein-SDS intermediate product, cause and carry a large amount of negative charge on testing protein to such an extent as to considerably beyond the quantity of electric charge of protein itself, so that the protein migration speed in running gel is only dependent upon the molecular weight of itself, thus ignore the difference in the migration rate that protein itself brings because electric charge is different.After gel electrophoresis, remove SDS with eluent, the activation recovering of testing protein.After hatching a period of time, testing protein is fully hydrolyzed substrate gelatin, then with coomassie brilliant blue staining, can show a white ribbon clearly.Density according to this white ribbon and area, just can measure the activity of enzyme[21]。
Quantitative fluorescence method is a kind of new method, can be with the activity of selective enumeration method Primary chondrocyte inner tissue E.C. 3.4.21.64.Its ultimate principle is as follows: at 5-nitrosalicylaldehyde (5-Nitrosalicylaldehyde, be called for short NSA) exist under conditions of, peptide substrate derivant (4-methoxy-β-naphthylamine by cell Yu cathepsin K to be measured, be called for short 4M β NA) jointly hatch a period of time after measure the light absorption value of its fluorescence-causing substance by fluorescence spectrophotometer, as control experiment value.After being subsequently adding the micromolecular inhibitor of cathepsin K, the absorption value of re-test fluorescence-causing substance.Finally, by add inhibitor and unchecked dose absorption value ratio calculation (Enzyme activities:Obtain the activity of cathepsin.
2. the condition of culture of cell
The cultivation of Primary chondrocyte uses DMEM culture medium (including 4.5g/L glucose, 10% hyclone, 1% Pen .-Strep, 1% glutamate, Glu, 1%HEPES buffer), cultivates 10 days continuously, system as a control group；Then same DMEM culture medium is used, but contain 50 μ g/mL ascorbic acid (Ascorbic acid simultaneously, and 7.5mM β-phosphoglycerol (β-glycerophosphate AA), β-GP), cultivate 10 days continuously, as experimental group system, it act as stimulating inducing cell to produce more cathepsin.Meanwhile, chondrocyte is also passed on by the present invention, uses DMEM culture medium (to include 4.5g/L Glucose, 10% hyclone, 100U/mL penicillin and 100 μ g/mL streptomycins, 1% glutamate, Glu, 1%HEPES buffer；Hereinafter pass on step and all use this culture medium) cultivate about 48h.After remove this culture medium, with PBS clean after, add 2mL pancreatin (Trypsin) be placed in 37 DEG C of incubators wait cell suspension, be subsequently adding 8mL DMEM in high glucose culture medium piping and druming mixing.Divide equally 5mL mixed liquor centrifugal (400g, 5min) in 15mL centrifuge tube, obtain lower sediment, and add 500 μ L Extraction buffer (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) standby to make.Remaining 5mL mixed liquor is placed in new culture dish, adds 4～5mL DMEM in high glucose culture medium and be placed in incubator (being labeled as Passage 1, P1), be used for passing on.(attached: the preservation of P1, and the most same above-mentioned steps of preservation passing on P2, P3).Accompanying drawing 10 is shown in chondrocyte Primary chondrocytes change of (primary, the first generation, the second filial generation, the third generation) in succeeding generations.
3. result and discussion
3.1 use by the chondrocyte (primary P0, first generation P1, second filial generation P2, third generation P3) before and after passing on, and utilize gelatin zymography to test the activity of cathepsin K therein respectively.Using DMSO as the matched group (A) of without inhibitor, experimental group is separately added into 1 μM of inhibitor (B:ODAN, C:CKI-E, D:CKI-F), and experimental result is shown in accompanying drawing 11.
Knowable to figure (A), primary P0 to second filial generation P2 gradually strengthen with algebraically for the activity of chondrocyte inner tissue E.C. 3.4.21.64, and expression is the highest when the second filial generation.When adding inhibitor ODAN (B), CKI-E (C), when CKI-F (D) and concentration reach 1 μM, the activity of cathepsin is the most substantially suppressed.
3.2 use by the chondrocyte (primary P0, first generation P1, second filial generation P2, third generation P3) before and after passing on, and utilize quantitative fluorescence method to test the activity of cathepsin K therein.Matched group with DMSO as without inhibitor, and it is separately added into the sample of 1 μM and 2 μMs inhibitor (ODAN, CKI-E, CKI-F) as experimental group, test result is shown in accompanying drawing 12.
Knowable to figure (A) result: gradually strengthen for the activity of chondrocyte inner tissue E.C. 3.4.21.64 from primary P0 to second filial generation P2, and expression is the highest when the second filial generation.Then when being separately added into 1 μM, after 2 μMs of inhibitor (ODAN, CKI-E, CKI-F), the depression effect of intracellular cathepsin K is strengthened by they with the rising of inhibitor concentration, sees figure (B).
3.3 use 50 μ g/mL ascorbic acid (Ascorbic acid, and 7.5mM β-phosphoglycerol (β-glycerophosphate AA), β-GP) continued stimulus induce 10 days chondrocyte and without stimulate induction chondrocyte as experiment contrast group, utilizing quantitative fluorescence method to detect the expression of intracellular cathepsin K, test result is shown in accompanying drawing 13.
Result (A) shows: when using without when stimulating the cell induced as controlled trial group, through stimulating the relative activity of the intracellular cathepsin K of induction to be increased to 140 by 100, i.e. the expression of cathepsin K substantially increases.Then, being sequentially added into the inhibitor (CKI-E, CKI-F, ODAN) of 0.1 μM or 1 μM, the depression effect of intracellular cathepsin K is all strengthened by they with the rising of inhibitor concentration, sees figure (B).
Test result indicate that of 3.4 comprehensive above gelatin zymography and quantitative fluorescence method: in the chondrocyte passed on, the expression of cathepsin K reaches maximum when P2；After adding inhibitor ODAN, CKI-E or CKI-F, the activity of they inner tissue the most capable of inhibiting cell E.C. 3.4.21.64s.And after chondrocyte is stimulated by induction, the expression of intracellular cathepsin K is significantly increased；Equally, should under the conditions of when add after ODAN, CKI-E or CKI-F them the most all can the activity of inhibiting cathepsin K.
Detailed description of the invention
Existing (±) E4 (para-/meta-) synthesis on the premise of[26]Take 0.61g parent compound (±) E4 (para-) and other the compound obtained from commercial channel: 0.43g to (±) methyl sulfone phenylboric acid ((4-(methylsulfonyl) phenyl) boronic acid), 65.3mg [1,1'-double (diphenylphosphine) ferrocene] palladium chloride (Pd (dppf) Cl2) and 0.5g potassium carbonate powder be separately added in the 100mL round-bottomed flask with stirring magneton, then the oxolane of 40mL is added in round-bottomed flask as reaction dissolvent, then injects 2mL water with liquid-transfering gun.After three times (nitrogen-vacuum) is filled and taken out reaction system, it is heated to reflux about 6h[27].And monitoring reaction process with thin layer chromatography (TLC), question response is complete, is evaporated by the oxolane in flask with Rotary Evaporators.It is subsequently adding 60mL ethyl acetate and 20mL water, separates organic facies with separatory funnel, then be extracted with ethyl acetate 3 aqueous phases, merge organic facies.And successively by water, saturated sodium bicarbonate and saturated sodium-chloride washing organic facies.Be dried with anhydrous sodium sulfate, to be dried complete, filter out this desiccant, be evaporated ethyl acetate solvent, obtain compound (±) crude product of CKI-E；Finally with petroleum ether: the mixed solvent of ethyl acetate=1:1 (volume: volume), as developing solvent, carries out silica gel column chromatography purification, (±) the white powdery solids 0.23g of CKI-E.
Same step, with (±) E4 (meta-) replace (±) E4 (para-), other experiment condition is identical, (±) the white powdery solids 0.19g of CKI-F.
CKI-E characterization result is 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): δ 174.48 (s), δ 145.85 (s), δ 140.32 (s), δ 139.28 (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), δ 40.59 (s), δ 30.64 (s), δ 25.67 (s), δ 22.71 (s), δ 22.40 (s).
MS(ESI)m/z:414.1841[M+H]+,827.3567[2M+H]+.
CKI-F characterization result is 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): δ 174.50 (s), δ 146.02 (s), δ 140.75 (s), δ 139.87 (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), δ 44.64 (s), δ 43.73 (s), δ 40.57 (s), δ 30.67 (s), δ 25.74 (s), δ 22.64 (s), δ 22.47 (s).
MS(ESI)m/z:414.1839[M+H]+,827.3571[2M+H]+.
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Claims (3)
1. a 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. the hydrazine nitrile cathepsin inhibitors described in claim 1 or 2 is in preparation treatment osteoarthritis drugs
Application.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436168A (en) * | 2000-06-14 | 2003-08-13 | 霍夫曼-拉罗奇有限公司 | Beta-amino acid nitrile derivs. |
| US20060122184A1 (en) * | 2002-12-05 | 2006-06-08 | Axys Pharmaceuticals, Inc. | Cyanomethyl derivatives as cysteine protease inhibitors |
| WO2009001129A1 (en) * | 2007-06-26 | 2008-12-31 | Astrazeneca Ab | 1-cyanocyclopropyl-derivatives as cathepsin k inhibitors |
| CN102731344A (en) * | 2012-06-01 | 2012-10-17 | 吉林大学 | Hydrazine nitrile cathepsin inhibitors with different P<3> structures, and application thereof |
| CN103086923A (en) * | 2013-01-21 | 2013-05-08 | 吉林大学 | Hydrazine cathepsin K inhibitor and application thereof in treating osteoporosis |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436168A (en) * | 2000-06-14 | 2003-08-13 | 霍夫曼-拉罗奇有限公司 | Beta-amino acid nitrile derivs. |
| US20060122184A1 (en) * | 2002-12-05 | 2006-06-08 | Axys Pharmaceuticals, Inc. | Cyanomethyl derivatives as cysteine protease inhibitors |
| WO2009001129A1 (en) * | 2007-06-26 | 2008-12-31 | Astrazeneca Ab | 1-cyanocyclopropyl-derivatives as cathepsin k inhibitors |
| CN102731344A (en) * | 2012-06-01 | 2012-10-17 | 吉林大学 | Hydrazine nitrile cathepsin inhibitors with different P<3> structures, and application thereof |
| CN103086923A (en) * | 2013-01-21 | 2013-05-08 | 吉林大学 | Hydrazine cathepsin K inhibitor and application thereof in treating osteoporosis |
Non-Patent Citations (1)
| Title |
|---|
| MAXIM FRIZLER等: "structural optimization of azadipeptide nitriles strongly increases association rates and allows the development of selective cathepsin inhibitors", 《J.MED.CHEM.》 * |
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