CN106963776A - A kind of application of compound in the disease medicine using cathepsin K as target is prepared - Google Patents

Abstract

The present invention relates to a kind of application of compound in the disease medicine using cathepsin K as target is prepared, the compound is cathepsin K nonactive site inhibitor, it acts on cathepsin nonactive site, with significantly inhibit collagen and elastomer protein degradation activity, activity without influenceing the other extracellular substrates of degraded, the disease being characterized available for preparation treatment with cathepsin K unconventionality expression or activation, including osteoporosis, gingivitis (gingivitis and periodontitis), osteitis deformans, metabolic bone disease, fracture, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, osteogenesis imperfecta, malignant hypercalcemia, Huppert's disease, metastatic osteopathy and pain.

Description

A kind of application of compound in the disease medicine using cathepsin K as target is prepared

Technical field

The present invention relates to cathepsin inhibitors technical field, specifically, it is related to a kind of group with high selectivity Knit application of the Proteinase K nonactive site inhibitor in disease medicine of the suppression using cathepsin K as target is prepared, the disease Disease includes osteoporosis, gingivitis (gingivitis and periodontitis), osteitis deformans, metabolic bone disease, fracture, rheumatoid joint Inflammation, osteoarthritis, periprosthetic osteolysis, osteogenesis imperfecta, malignant hypercalcemia, Huppert's disease, metastatic osteopathy and Pain.

Background technology

It is related to the related illness of abnormal bone resorption a lot, including but not limited to osteoporosis, gingivitis (gingivitis and tooth Zhou Yan), osteitis deformans, metabolic bone disease, fracture, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, into Bone is not complete, malignant hypercalcemia, Huppert's disease, metastatic osteopathy and pain.One of most common is osteoporosis, and its is common In the elderly and postmenopausal women.In the China crowd of more than 50 years old, there is 30% to suffer from osteoporosis, as the average life span prolongs Long, aging population trend is serious, and the incidence of disease more and more higher of osteoporosis, the fracture thus triggered causes the disease of gerontal patient Dead rate and disability rate increase [Luo Xianzheng, the Epidemiology of osteoporosis, Chinese Rural medicine, 17 (2):1651- 1662,2010]。

Osteoporosis is that the relation between the bone information of osteoclast and the bon e formation of Gegenbaur's cell is unbalance caused.Group Knitting Proteinase K, optionally great expression is in osteoclast, and content reaches its physiological action substrate exactly in organic bone matrix 95% NTx, it by complete triple helix body structure collagen degradation into irregular structure fibre debris, except I Outside Collagen Type VI, cathepsin K can also degrade osteopontin and osteonectin in bone matrix, be bone resorption activity in osteoclast Most strong key enzyme.The variation of human cathepsin's K gene expressions can cause pycnodysostosis, ultimately result in sclerotin The increase of loose and bone fragility.Bone can not be formed using the in vitro osteoclast of cathepsin K gene depleted mice marrow culture Absorption lacuna, it is impossible to type i collagen of effectively degrading.

Cathepsin K is the key target of medicine for treating osteoporosis, and preclinical and clinical data shows histone Enzyme K inhibitor can reach 80% to the inhibitory action of bone information, the Biological indicators without reducing bon e formation.Based on this preventing and treating At least two kinds of clinical tests failure in 4 kinds of cathepsin K inhibitors of osteoporosis strategy, failure cause is with causing non-bone The fibrosis of bone tissue is relevant.Such inhibitor is the not only inhibition of histone using Cathepsin K activities site as target spot Collagenase activity related to drug effect enzyme K, while also other physiologically actives of inhibitory enzyme.Research finds cathepsin K defect Mouse shows lung airway damage in addition to phenotype expected from performance skeletal system, also, it is easy to lung and skin is occurred fibrosis, first shape Gland globulin (Tg) discharges thyroxin obstacle, even results in learning and memory defect etc., shows that active site inhibitor can Side effect can be caused, this proposes challenge to develop new osteosporosis resistant medicament.

CatK inhibitor patents are active site inhibitor both at home and abroad at present, not yet there is nonactive site inhibitor.

The content of the invention

One aspect of the present invention is that there is provided one kind to specifically bind cathepsin K for deficiency of the prior art non- Avtive spot, the material of selective depression Cathepsin K activities is in the disease medicine using cathepsin K as target is prepared Using.

In order to solve the above technical problems, the technical scheme that the present invention takes is as follows：

A kind of cathepsin K nonactive site inhibitor is in the disease medicine using cathepsin K as target is prepared Using；The cathepsin K nonactive site inhibitor is selected from：Chemical formula parent nucleus is tricyclic structure shown in I, II or III Compound, pharmaceutically useful ester, acid amides or the salt of the compound, and salt or composition comprising the ester or acid amides；

Wherein：C rings are selected from phenyl ring and cyclohexyl ring；There are contraposition and meta diketone or five rings and six cyclic lactone class knots in A rings Structure；The bit substituent of A rings 1,2 is selected from furan nucleus, pyranoid ring, dihydro substituted furan ring and dihydro substituted pyrane ring.

As the preference of the present invention, C rings are selected from methyl phenyl ring and dimethyleyelohexane basic ring.

As another preference of the present invention, the cathepsin K nonactive site inhibitor is selected from A rings or C rings Ester, acid amides and salt that substituent is formed.

As another preference of the present invention, the cathepsin K nonactive site inhibitor is tanshinone IIA sulfonic acid Sodium.

As another preference of the present invention, the disease using cathepsin K as target is selected from osteoporosis, gum Disease, osteitis deformans, metabolic bone disease, fracture, rheumatoid arthritis, osteoarthritis, periprosthetic osteolysis, skeletonization are not Entirely, malignant hypercalcemia, Huppert's disease, metastatic osteopathy and pain；The gingivitis is selected from gingivitis and periodontitis.

As another preference of the present invention, the disease using cathepsin K as target is bone information abnormal diseases.

As another preference of the present invention, the bone information abnormal diseases are osteoporosis.

Another aspect of the present invention provides a kind of pharmaceutical composition in the disease medicine using cathepsin K as target is prepared Application, described pharmaceutical composition include cathepsin K nonactive site inhibitor, the cathepsin K non-active site Point inhibitor is selected from：Chemical formula parent nucleus be tricyclic structure shown in I, II or III compound, the compound it is pharmaceutically useful Ester, acid amides or salt, and salt or composition comprising the ester or acid amides；

Wherein：C rings are selected from phenyl ring and cyclohexyl ring；There are contraposition and meta diketone or five rings and six cyclic lactone class knots in A rings Structure；The bit substituent of A rings 1,2 is selected from furan nucleus, pyranoid ring, dihydro substituted furan ring and dihydro substituted pyrane ring.

As the preference of the present invention, described pharmaceutical composition is suppressed with the cathepsin K nonactive site Agent is sole active agent.

As another preference of the present invention, the disease using cathepsin K as target is bone information abnormal diseases.

The invention has the advantages that：

The cathepsin K nonactive site inhibitor of the present invention acts on human cathepsin K amino acid sequences (1： Positioned at amino acid sequence Tyr⁸⁷To Gly¹⁰², 2：Gly¹⁰⁸To Glu¹¹⁸), pharmacodynamic study shows, can significantly inhibit human osteoclast Activity, improves estrogen loss removal ovary osteoporosis Mouse Bone architectural feature and physiological status, including improve bone density, improve Biomethanics of bone fine structure and raising bone tissue etc., so that with significant function of resisting osteoporosis, and to brain learning Memory capability is without influence.Such inhibitor provides new thinking for osteosporosis resistant medicament research and development.

Brief description of the drawings

Fig. 1：T06 is used as non-active site point feature.(A) SDS-PAGE analyzes type i collagen degraded, (B) collagen degradation IC50 schemes, and (C) SDS-PAGE analyzes the degrading activity of gelatin, and (D) SEM observations T06 has the type i collagen fiber with the absence of Hydroxy-proline is quantitative determined after electron microscope, (E) type i collagen Fiber Digestion, and T06 docking site is identified in (F) molecular docking.

Fig. 2：T06 and T02 is to osteoclast TRACP positive cells and resorption pit of osteoclast in vitro (A, C) and osteoclastic The influence (B, D) of cell metabolic activity and cell number.

Fig. 3：The changes of weight (A) and organ coefficient (B) of mouse modeling 10 weeks.

Fig. 4：T06 includes to uterus area (C), uterus cavity area (D), and son to Uterine coefficient (A) and uterus sections (B) The influence of uterine cavity epithelial cell thickness (E).

Fig. 5：Influences of the T06 to ovariectomized mouse femur (A) and vertebrae L5 (B) bone density.

Fig. 6：Influence to mouse bone information associated serum biochemistry RANKL and CTX-I.

Fig. 7：T06 is to mouse behavioral implications.(A) spacious field is tested, (B) Elevated plus-maze, and (C) water maze is instructed for the 2-5 days Practice, (D) water maze is tested on the 6th day.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.These embodiments are interpreted as being merely to illustrate this hair It is bright rather than limit the scope of the invention.After the content of the invention recorded has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalence changes and modification equally fall into the model that the claims in the present invention are limited Enclose.

, there is inseparable relation the problem of osteoporosis with Cathepsin K activities in osteoclast, therefore can press down The compound of Cathepsin K activities processed, also implies that the bone loss and fracture phenomenon that can improve caused by osteoporosis.

A kind of cathepsin K nonactive site inhibitor (Cathepsin K exosite of the present invention Inhibitor), the compound mother nucleus structure is selected from one of the following：

Preferably：C rings are methyl phenyl ring or dimethyleyelohexane basic ring；There is contraposition or meta diketone, 1,2 substitution in A rings Base is furan nucleus and dihydro substituted furan ring.The selection of above-mentioned A ring and C rings can be selected simultaneously or separately.The invention discloses compared with Good structure is tanshinone IIA sodium sulfonate, but is not limited thereto.Wherein tanshinone IIA sodium sulfonate structural formula is：

The invention discloses a kind of purposes of cathepsin K nonactive site inhibitor, applied to preparing anti-osteoporosis Medicine.It is tanshinone IIA sodium sulfonate the invention discloses preferred construction, but is not limited thereto.Below by way of experiment specifically The bright present invention is applied to the drug effect for the treatment of osteoporosis.

Embodiment 1：The screening of novel tissue Proteinase K nonactive site inhibitor and confirmation

To 7 monomeric compounds in the red sage root, I type structure danshenxinkun D, II type structure dihydrotanshinone Is, Tanshinone I is red Join ketone IIA, Cryptotanshinone, tanshinone IIA sodium sulfonate and the hidden spirolactone of the type III structural table red sage root have carried out nonactive site suppression The screening of agent.Structure is as follows：

1 experiment material

1.1 medicinal materials and reagent

Monomeric compound danshenxinkun D, dihydrotanshinone I, Tanshinone I, tanshinone IIA, Cryptotanshinone, tanshinone IIA Sodium sulfonate and the hidden spirolactone of the table red sage root, buy from chemfaces companies；Benzyloxycarbonyl-Phe-Arg-7-amido- 4-methylcoumarin (Z-FR-MCA) is purchased from WAKO companies of Japan；Chondroitin sulfate A (CSA) (C4-S), E-64 (L-3- Carboxy-trans-2-3-epoxypropionyl-leucylamido- (4guanidino)-butane) and pepsin purchase From Sigma Co., USA；Type i collagen is purchased from Affymetrix companies of the U.S.；Micro porous filtration filter-membrane centrifugal tube is purchased from Amico Millipore companies.

2 experimental methods

2.1 collagenous fibres degrading activities

Using gel electrophoresis (SDS-PAGE), soluble Type I collagenolysis in 100mM sodium-acetate buffers (pH5.5, Include 2.5mM DTT and 2.5mM EDTA) in, ultimate density is 0.6mg/ml, successively by CatK (ultimate density 400nM), C4- S (ultimate density 200nM) and compound monomer (various concentrations inhibitor) are added in the reaction solution containing I-type collagen, It is 50 μ L to make overall reaction liquid capacity.Mix, 28 DEG C are incubated 4 hours, and 1 μ L 100 μM of E64 terminating reactions are added after taking-up.Using 10% PAGE gel electrophoretic separation, Camas light blue is dyed 20 minutes, with acetate-methanol (4:1) decolourize.Type i collagen The bands of α 1 gray scale with GeneSnap (SyngeneInc.Frederick, MD) software carry out quantitative analysis.Nonactive site The bands of α 1 degraded of type i collagen can be suppressed with active site inhibitor.

2.2 elastomer degrading activities

The Congo red elastomer albumen of 1mg (Congo-Red elastin) is weighed, the 100mM sodium acetates for being placed in 100 μ L delay In fliud flushing (pH5.5 includes 2.5mM DTT and 2.5mM EDTA), the CatK and various concentrations for adding final concentration of 1 μM suppress Agent, 37 DEG C, 200 turns/min shaking tables are incubated 16 hours.Sample takes out, 8000 turns/min, centrifuges 5min, takes the μ L of supernatant 90, Fluorescence signal is detected under 490nm wavelength.Calculation formula：Inhibiting rate %=100- (1-Vi/V0).Vi and V0 represent exist respectively With in the absence of the fluorescence signal in the case of inhibitor.

2.3 Z-FR-MCA Binding Capacities (avtive spot)

Inhibitor is diluted to concentration with 100mM sodium-acetate buffers (pH5.5 includes 2.5mM DTT and 2.5mM EDTA) For 25 μM, 96 orifice plates are added, end reaction capacity is 1mL.The CatK for adding final concentration of 5nM is incubated 5 minutes, adds 5 μ L bottoms Thing 1mM/mL Z-FR-MCA solution starts reaction, detection fluorescence signal (diverging optical wavelength 460nm, absorb optical wavelength 355nm). Experiment sets negative control group (no inhibitor), positive controls (E-64, active site inhibitor).Calculation formula：Inhibiting rate % =100- (1-Vi/V0).Vi and V0 represent to exist respectively and in the absence of the fluorescence signal in the case of inhibitor.

2.4 gelatin degradations (avtive spot)

Using gel electrophoresis (SDS-PAGE), soluble Type I collagen obtains gelatin in 20 minutes in 95 DEG C of heating, is dissolved in 100mM sodium-acetate buffers (pH5.5 includes 2.5mM DTT and 2.5mM EDTA), ultimate density is 1.8mg/ml, by CatK (ultimate density 5nM) and monomeric compound (25 μM of ultimate density) are added in reaction solution, make the μ L of overall reaction liquid capacity 50.Mix, 28 DEG C are incubated 4 hours, and 1 μ L 100 μM of E64 terminating reactions are added after taking-up.Using 10% PAGE gel electrophoresis point From Camas light blue is dyed 20 minutes, with acetate-methanol (4:1) decolourize.The gray scale GeneSnap of the bands of α 1 of gelatin (SyngeneInc.Frederick, MD) software carries out quantitative analysis.Active site inhibitor, the bands of gelatin α 1 will be suppressed, Nonactive site inhibitor is acted on gelatin degradation unrestraint.

2.5 molecular docking

Two nonactive sites of computer simulation inhibitor and CatK are docked, and further confirmation binding site and combination are special Property.

2.6 collagenous fibres degrade (SEM Electronic Speculum)

SEM Electronic Speculum is used for observing difference of the collagenous fibres in CatK patients before and after intervention.1mg mouse type i collagen fibers and 1 μM CatK is incubated in 37 DEG C of shaking tables jointly, and concussion is stayed overnight.The collagenous fibres being degraded are in SEM ESEMs (Helios NanoLab 650 FEI) under observe metamorphosis.

3 experimental results

7 red sage root compounds to selection carry out Cathepsin K activities screening, and cathepsin K inhibitor has suppression Glue fibrillin and elastomer protein degradation activity, Z-FR-MCA and gelatin degradation activity are active site inhibitors Synthesis and physiologic substrate, for initial screening active site inhibitor.

The Cathepsin K activities of different compounds in the red sage root of table 1

^*Dihydrotanshinone I and tanshinone IIA sodium sulfonate are compared with Odanacatib respectively, P ＜ 0.05；^#Dihydrotanshinone I Compared with tanshinone IIA sodium sulfonate, P ＜ 0.05.

As a result (table 1) is found, tanshinone IIA sodium sulfonate has most strong collagenous fibres degrading activity (IC₅₀For 2.7 ± 0.2 μM) and elastomer degrading activity (IC₅₀For 6.8 ± 1.4 μM), optimal close to current clinical effectiveness, ground in III phase clinics The cathepsin K inhibitor Odanacatib studied carefully, while tanshinone IIA sodium sulfonate does not show Z-FR-MCA activity and gelatin drop Solution activity (table 1 and Fig. 1 in A-C), it is nonactive site inhibitor to show tanshinone IIA sodium sulfonate.Electric Microscopic observation is sent out simultaneously Existing tanshinone IIA sodium sulfonate inhibits digestion of the CatK to collagenous fibres, and hydroxy-proline quantitative determination demonstrates the result (figure D-E in 1).The combination for finding tanshinone IIA sodium sulfonate and human cathepsin K, optimal combination side are tested using molecular docking Formula is shown in F in Fig. 1, and it is that Met97 and Glu94, Tyr87 are combined with sulfonate group to form hydrogen bonding sites.

The collagenous fibres degrading activity of dihydrotanshinone I is only second to tanshinone IIA sodium sulfonate, elastomer in II type structures Degrading activity and tanshinone IIA sodium sulfonate are without significant difference, but display simultaneously is lived with certain Z-FR-MCA and gelatin degradation Property, there were significant differences with tanshinone IIA sodium sulfonate, it is possible to dihydrotanshinone I simultaneously with avtive spot and nonactive site knot Close.The elastomer degrading activity of Tanshinone I and tanshinone IIA is better than collagen degrading activity, shows that certain Z-FR-MCA lives Property.The danshenxinkun D of I type structures and the hidden spirolactone collagenous fibres degraded of the table red sage root of type III structure and elastomer are degraded and lived Property is not notable, and shows certain Z-FR-MCA and gelatin degradation activity simultaneously.

Embodiment 2：Influences of the cathepsin K inhibitor T06 to osteoclastic bone resorption

Enzyme system in the endochylema of osteoclast expressed in abundance, breaks up in osteoclast precursor cells to mature osteoclast During there is a series of significant protein to produce, identification that can be as osteoclast and its mark of differential period.Its In, Tartrate resistant acid phosphatase (the tartrate-resistant acid expressed by the osteoclast precursor cells after differentiation Phosphatase, TRAP) be considered as osteoclast marker enzyme.This experiment induces osteoclast model using marrow, investigates T02, T06 break up to osteoclast, the influence of TRAP activity.

1 experiment material

1.1 medicinal materials and reagent

Animal：The brood SD mouse 5 of newborn 1-2 days, male and female are not limited, body weight 7-8g, are tested by The 2nd Army Medical College dynamic Thing center is provided.

Tanshinone IIA sodium sulfonate：Diluted when using with deionized water dissolving；RANKL, M-SCF are public purchased from U.S. Sigma Department.

2 experimental methods

2.1 Osteoclast culture

Newborn 3 days SD mouse tibias are taken, culture medium rinses ossis to collect bone marrow cell, adds equivalent Ficoll examinations Agent separates myelomonocyte, with containing 25ng/mL M-CSF, 25ng/mL RANKL cell factor and 10% hyclone α-MEM culture mediums cultivated, change within every 3 days osteoclast differentiation and maturation after liquid 1 time, 6 days.

2.2 osteoclast activity researchs

(1) osteoclast TRACP activity；Mature osteoclast is with 1 × 10⁵/ mL is inoculated in 96 orifice plates, is changed plus different dense T06 (0.1 .0.5,1.0,10.0 μM) is spent, negative control group adds the osteoclast inducing culture of not drug containing, cultivates 48h Afterwards, the μ L of culture medium 20 of removal are taken, according to TRACP kit spectrophotometry osteoclast activities.Determine TRAP work Property, standard curve, the nmol for the p-nitrophenol that TRAP activity is generated with the osteoclast per hole are made with p-nitrophenyl phenol solution Number is represented.

(2) osteoclast vigor；After cell maturation, with 1 × 10⁵/ mL is inoculated in 96 orifice plates, and bulls bone bone is inserted in advance Piece, adds 1 μM of T06 with potential suppression CatK activity.Continue to cultivate after 48h, take 100 μ L culture mediums, add 20 μ L CellTriter-blue reagents, gently shake 10s and mix, 37 DEG C of incubation 30min, sepectrophotofluorometer detection (diverging optical wavelength 560nm, absorbs optical wavelength 590nm).Selection does not have virose concentration range to carry out osteoclast activity test to cell viability.

(3) osteoclast number and TRACP positive cell numbers；Mature osteoclast is with 1 × 10⁵/ mL is inoculated in 96 holes In plate, 1 μM of T06 osteoclast inducing culture is added, is cultivated in advance after 48h, culture medium is removed to be measured to another orifice plate TRACP activity.According to TRACP staining kits to osteoclast nuclear targeting, TRACP positive osteoclasts numbers are calculated.

3 experimental results

This experiment investigates cathepsin K suppression using bone marrow cell and Gegenbaur's cell co-culturing, inducing osteoclast model Preparation breaks up and the active effects of TRAP to osteoclast, as shown in table 2.Bone marrow cell is developed after 8 days through Fiber differentiation to be into Ripe osteoclast, after acting on 48h through T06 and 10 of 1-10 μM μM of T02 medicine, Tartrate resistant acid phosphatase (TRAP) is living Property suppress (P ＜ 0.05) by obvious.

The T06 of table 2 to Myelogenic Osteo influence (n=10,)

* P ＜ 0.05, * * P ＜ 0.01vs Control.

Tanshinone IIA sodium sulfonate completely inhibits A in the bone information of osteoclast, Fig. 2 at 1 μM and shows TRACP dyeing The Bone resoiption pit of osteoclast and Toluidine blue staining.In control group, it can be found that longer and deep bone trace and bone are fallen into Nest, 1 μM of T06 group only has smaller and shallow bone lacuna, it was demonstrated that 1 μM of tanshinone IIA sodium sulfonate can substantially reduce bone information surface. However, the metabolic activity determination experiment of osteoclast is found, medicine does not have toxic action to osteoclast, it is impossible to substantially reduce The number (B in Fig. 2) of TRACP stained positive osteoclasts.C-D shows dihydrotanshinone I to osteoclast metabolic activity in Fig. 2 Do not make significant difference, TRACP positive cell numbers are not made significant difference, but there is certain suppression to make to bone information surface area With.Cell experiment proves that tanshinone IIA sodium sulfonate is a preferable anti-bone information medicine.

Embodiment 3：Influences of the cathepsin K inhibitor T06 to removal ovary osteoporosis mouse

1 experiment material

Animal：C57BL6 mouse are provided by University of British Columbia experimental animal.Animal sub-cage rearing (3~4 Only/cage) in air-conditioning greenhouse, 21 ± 2 DEG C of temperature, humidity 40~60%；Fed with pellet, free water.

2 experimental methods

Animal packet：By female C57BL6 mouse 30 (18-22g), 3 groups are randomly divided into by body weight, every group 10, including Sham-operation group (SHAM, gavage physiological saline 10ml/kg), osteoporosis model group (OVX, to physiological saline 10ml/kg) and T06 (40mg/kg/d) 3 groups.After recovering 5 days, continuous gavage is administered 12 weeks.

Modeling method：Isoflurane anesthesia on operating table, the otch of two 0.5cm sizes is cut at back, and separating muscle is cut Bilateral ovaries are taken out after opening.Sham-operation group is cut will be without any processing behind back, sewing-up cut, sterilization；Operation group is by bilateral Cut off after ovary ligation, sewing-up cut, sterilization.

Observation index：

(1) body weight：Body weight of mouse is weighed weekly.

(2) organ coefficient：After mouse is put to death, its heart, liver, spleen, lung, kidney and the affiliated group in uterus and fat are peeled off rapidly Fat, is weighed immediately.Organ coefficient formula：Organ coefficient=organ weights (mg)/body weight (g), to uterus sections observation.

(3) serum TGF-β, CTX-I and RANKL

Eye socket takes blood, is placed in anticoagulant sodium heparin pipe, is centrifuged 10 minutes with 3000 turns/min, separates serum, freezes in -80 It is DEG C standby.According to kit method detection.

(4) bone density and bone histomorphometric

After mouse is put to death, left femur is peeled off, affiliated group around bone is rejected, is scrubbed with physiological saline, use tinfoil paper Paper bag is good, and -80 DEG C freeze, and bone tissue parameter is detected for μ CT.

(5) behaviouristics and mnemonic learning ability

Mouse carries out spacious field experiment, Elevated plus-maze and water maze laboratory, for evaluating the general of mouse before execution Behavior and ability of learning and memory.

3 experimental results

The influence of 3.1 pairs of body weight and Main Organ Coefficients

As shown in table 3, after modeling first week, OVX and OVX+T06 body weight are just significantly higher than normal Sham groups, modeling knot Shu Hou, Ovariectomy model group mouse weight value added is apparently higher than Sham groups, the T06 body weight evolution nothing compared with model group Significant difference.

As shown in B in Fig. 3, each Main Organ Coefficients of Ovariectomy model group mouse (heart, liver, spleen, lung, kidney) are significantly lower than Sham groups, OVX and OVX+T06 compare Main Organ Coefficients (heart, liver, spleen, lung, kidney) without conspicuousness change.

The influence in 3.2 pairs of uterus

After modeling 12 weeks, take out uterus and weigh, the Mouse Uterus atrophy of discovery OVX and OVX+T06 groups, no significant difference, Two groups of Uterine coefficients are substantially less than Sham groups, it is shown that the obvious uterus hyperplasia side effects of T06.

Uterus sections observation is found, OVX and OVX+T06 group Mouse Uterus chambers are reduced significantly, and uterus area is substantially reduced, Uterine cavity epithelial cell thickness is substantially reduced.After removal ovary, estrogen loss causes developing womb to be obstructed, and atrophy, uterus occurs Epithelial cell structure becomes close, and uterine cavity epithelial cell is not obvious, occurs being broken.

3.3 pairs of bone densities and bone condention Index Influence

Research is found, after μ CT scan bones, and the bone density (BMD) of L5 sections of OVX groups femur and vertebrae significantly drops Low (P ＜ 0.05).OVX+T06 is compared OVX groups, and femoral bmd is significantly improved (P ＜ 0.05), and L5 bone densities, which have, necessarily to be changed It is kind, no significant difference (Fig. 5).

The result of table 3 is shown, is compared with normal group, and OVX groups are shown, femur BV/TV ratios (BV/TV%) reduction by 48%, Bone trabecula number (Tb.N) reduction by 43%, bone trabecula thickness (Tb.Th) reduction by 8%, bone trabecula gap (Tb.Sp) increase 50%, Bone Connection Density (Conn.Dn) is reduced to 57%.Vertebrae L5, similar to femur, BV/TV ratios significantly reduce (P ＜ 0.001), bone trabecula number (Tb.N) is significantly reduced (P ＜ 0.05), and bone trabecula gap (Tb.Sp) significantly increases (P ＜ 0.05), Bone Connection Density (Conn.Dn) is significantly reduced (P ＜ 0.01).

Relative to Ovariectomy model group (OVX), OVX+T06 femur index, BV/TV ratios significantly increase (P ＜ 0.001), bone trabecula number (Tb.N) significantly increases (P ＜ 0.05), bone trabecula thickness increase (P ＜ 0.05), bone trabecula gap (Tb.Sp) it is substantially reduced (P ＜ 0.05), bone Connection Density (Conn.Dn) is significantly increased (P ＜ 0.01).L5 sections of vertebrae each Index is without significant changes.

Influences of the T06 of table 3 to femur and vertebrae L5 ovariectomized mouse bone measuring parameters

The influence of 3.4 pairs of Biochemical Indices In Serums

RANKL participates in osteoclast formation, differentiation, fusion, existence, activation, ripe overall process, OPG Reverse transcriptases RANKL is combined with the acceptor RANK on preosteoclast, the reduction of OPG/RANKL ratios, will suppress bone information.CTX-I is I type glue Former final catabolite, directly reflects the degraded state of ossein.Fig. 6 results are shown, are compared with OVX groups, blood in OVX+T06 Clear RANKL (P ＜ 0.001) and phosphorus CTX-I levels are significantly reduced (P ＜ 0.05).Illustrate that T06 has compared with high inhibition bone information to make With.

The influence of 3.5 pairs of behaviouristics and ability of learning and memory

Three groups of mouse carry out spacious field experiments, and the total activity distance of 5 minutes, average speed illustrates mouse work without significant difference Kinetic force is essentially identical.Compared with sham groups, mouse explores the time of interior zone significantly less (P ＜ 0.01).Elevated plus Labyrinth shows that three groups of mouse are opening the residence time of arm and closed arm and going out indegree without significantly before and after modeling Difference.Water maze laboratory is shown, is trained 2-5 days, OVX and OVX+T06 mouse search out the time required for water-bed platform daily Reduce, but the distance of swimming is shorter and shorter, illustrates that mouse obtains certain ability of learning and memory.6th day, OVX and OVX+T06 Group mouse is by the time between the number of times of negative platform and platform location without significant difference.Three behaviors model experiment results Show that OVX and the equal indifferences of OVX+T06 illustrate that CatK inhibitor T06 has no influence to OVX ability of learning and memory in mice.

Conclusion

We obtain nonactive site inhibitor tanshinone IIA sodium sulfonate by high flux screening, have investigated T02 and T06 Effect to osteoclastic bone resorption activity (osteoclast development quantity and TRAP activity), as a result finds the inhibitor in vitro Suppress bone resorption with preferably external.Meanwhile, we investigate anti-sclerotin using removal ovary osteoporosis mouse model Fluffing action, as a result compounds on estrogen missing osteoporosis is with preferably activity, and to mouse weight and each organ Coefficient finds that no estrogen sample is acted on to Uterine coefficient and section research, not found as Estrogen therapy causes body without influence The side effect such as reduction, uterus hyperplasia again, the side effect for not occurring ability of learning and memory missing caused by CatK missings.Therefore, I Believe nonactive site inhibitor tanshinone IIA sodium sulfonate have be further developed as osteosporosis resistant medicament potentiality and should Use prospect.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as Protection scope of the present invention.

Claims (10)

1. a kind of cathepsin K nonactive site inhibitor answering in the disease medicine using cathepsin K as target is prepared With；The cathepsin K nonactive site inhibitor is selected from：Chemical formula parent nucleus is the change of tricyclic structure shown in I, II or III Compound, pharmaceutically useful ester, acid amides or the salt of the compound, and salt or composition comprising the ester or acid amides；

Wherein：C rings are selected from phenyl ring and cyclohexyl ring；There are contraposition and meta diketone or five rings and six cyclic lactone class formations in A rings；A The bit substituent of ring 1,2 is selected from furan nucleus, pyranoid ring, dihydro substituted furan ring and dihydro substituted pyrane ring.

2. application according to claim 1, it is characterised in that C rings are selected from methyl phenyl ring and dimethyleyelohexane basic ring.

3. application according to claim 1 or 2, it is characterised in that the cathepsin K nonactive site inhibitor choosing Ester, acid amides and the salt formed from A rings or C ring substituents.

4. application according to claim 1, it is characterised in that the cathepsin K nonactive site inhibitor is pellet Join ketone IIA sodium sulfonates.

5. application according to claim 1, it is characterised in that the disease using cathepsin K as target is selected from bone Matter is loose, gingivitis, osteitis deformans, metabolic bone disease, fracture, rheumatoid arthritis, osteoarthritis, periprosthetic bone Dissolving, osteogenesis imperfecta, malignant hypercalcemia, Huppert's disease, metastatic osteopathy and pain；The gingivitis is selected from gingivitis And periodontitis.

6. application according to claim 1, it is characterised in that the disease using cathepsin K as target is that bone is inhaled Receive abnormal diseases.

7. application according to claim 6, it is characterised in that the bone information abnormal diseases are osteoporosis.

8. a kind of application of pharmaceutical composition in the disease medicine using cathepsin K as target is prepared, described pharmaceutical composition Comprising cathepsin K nonactive site inhibitor, the cathepsin K nonactive site inhibitor is selected from：Chemical formula is female Core is the compound of tricyclic structure shown in I, II or III, pharmaceutically useful ester, acid amides or the salt of the compound, and comprising described The salt or composition of ester or acid amides；

Wherein：C rings are selected from phenyl ring and cyclohexyl ring；There are contraposition and meta diketone or five rings and six cyclic lactone class formations in A rings；A The bit substituent of ring 1,2 is selected from furan nucleus, pyranoid ring, dihydro substituted furan ring and dihydro substituted pyrane ring.

9. application according to claim 8, it is characterised in that described pharmaceutical composition is with the cathepsin K non-live Property site inhibitor be sole active agent.

10. application according to claim 8, it is characterised in that the disease using cathepsin K as target is that bone is inhaled Receive abnormal diseases.