CN102167720B - Betulonic acid derivant for restraining differentiation of osteoclast, preparation method thereof and application thereof - Google Patents
Betulonic acid derivant for restraining differentiation of osteoclast, preparation method thereof and application thereof Download PDFInfo
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- CN102167720B CN102167720B CN2011100596092A CN201110059609A CN102167720B CN 102167720 B CN102167720 B CN 102167720B CN 2011100596092 A CN2011100596092 A CN 2011100596092A CN 201110059609 A CN201110059609 A CN 201110059609A CN 102167720 B CN102167720 B CN 102167720B
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Abstract
The invention relates to a betulonic acid derivant for restraining the differentiation of osteoclast, a preparation method thereof and application thereof. The preparation method comprises the steps of adding betulonic acid and di-brominated mair multi-yeast acid into absolute ether, stirring for 4h at a room temperature, and purifying; and adding thioacetamide and absolute ethyl alcohol, reacting in a reflowing way for 12h, concentrating and extracting reaction liquor, merging an organic layer, washing by saturated saline water, evaporating off an organic solvent, and purifying a coarse product by means of silicagel column chromatography to obtain the betulonic acid derivant I. By improving the structure of natural product betulonic acid and introducing a benzo heterocyclic ring into a C2 site and a C3 site, the method is used for effectively synthesizing the novel betulonic acid derivan, and the synthesis method is simple and convenient; and by improving the structure of the betulonic acid, the function of the compounds for restraining the differentiation activity of the osteoclast is obviously improved.
Description
Technical field
The present invention relates to a kind of suppress osteocyte differentiation betulonic acid verivate and preparation and application, belong to medicine and preparation thereof and applied technical field.
Background technology
Osteoporosis (Osteoporosis; OP) be a kind of health problem relevant with the age; Reduce with bone micro-structure to deteriorate to characteristic with the bone amount, cause the fragility increase of bone and be easy to fracture a kind of multifactor due to chronic systemic osteopathy (Sambrook P.et al.Lancet, 2006; 367,2010).Along with the aging of human society, the osteoporosis sickness rate constantly increases, and according to statistics, China's sufferers of osteoporosis face in 1999 is 1,900 ten thousand, and expecting the year two thousand fifty will be above 200,000,000.The women is more than the male sex for this disease, postmenopausal women especially, and osteoporosis and fracture complication sickness rate are higher, and about 1: 8 of ratio of males and females has a strong impact on the elderly, especially the quality of life of elderly woman.
The metabolism of bone is kept by ingenious balance between the bone forming of the bone resorption of osteoclast (Osteoclast) mediation and scleroblast (Osteoblast) mediation, and this balance is in case abnormal bone metabolism will take place destruction.When the activity of osteoclast far surpassed osteoblastic activity, the bone amount reduced, thereby caused the generation of osteoporosis; Otherwise, if scleroblast hyperactivity then can cause osteopetrosis (Boyle W.J.et al.Nature, 2003,423,337; Teitebaum S.L.et al.Science, 2000,289,1504).The metabolism of bone is divided into three steps: i.e. the absorption of bone, the formation of bone and the mineralising of ground substance of bone.In fact osteoporosis is exactly the metabolic unbalance result of bone, and therefore, the medicine of treatment osteoporosis also can be divided three classes according to its effect: promptly suppress bone resorption, promote bone forming and short bone mineralising.The medicine of inhibition bone resorption comprises oestrogenic hormon, thyrocalcitonin, diphosphonate, selective estrogen receptor modulators etc., and mainly osteoclast forms or the activity of inhibition osteoclast suppresses bone resorption and slow down bone calcium to lose through suppressing.Short osteoplastic medicine comprises parathyroid hormone, fluorochemical, statins, cell controlling elements etc., and this type of medicine ability stimulating osteoblast is active, makes the timely mineralising skeletonization of freshman bone tissue, lowers bone fragility, bone density improving and bone amount.Short bone mineralising medicine is mainly calcium agent and activated vitamin D (VD, alfacalcidol, U-32070E, CALCITRIOL USP, Parterol), is the basic medication of Prevention and Treatment of Osteoporosis.More than the use of these medicines some limitation and spinoff are all arranged, as: estrogenic use can increase danger such as mammary cancer, apoplexy, thrombus, therefore can not life-time service; Diphosphonate is unfavorable for absorbing, and produces gastrointestinal discomfort through regular meeting; Parathyroid hormone can not be oral, and life-time service may produce osteosarcoma, generally grows most and recommend be no more than 2 years duration of service.Therefore press for the new drug that people develop new, more effective, safer prevention and treatment osteoporosis.
So far, osteoporotic treatment is many to be target spot with the osteoclast, can be divided into the sophisticated osteoclast of multinuclear through the inhibition osteoclast precursor and reduce the bone degradation rate, improves bone resorption.Therefore; The bone resorption inhibitor that suppresses the osteoclast differentiation is the osteoporotic focus of treatment; Become the focus of drugmaker and each big pharmaceutical factory concern just gradually, this type medicine is expected to become the osteoporotic line medicine of treatment and substitute existing clinical application in the coming years.Is the monoclonal antibody of osteoclast differentiation factor RANKL like European's medication council (CHMP) in the Di Nuosaimai (Denosumab) of the Amgen (Amgen) of in December, 2009 approval, can with the RANKL effect, be neutralized into osteocyte excretory RANKL; The differentiation, the maturation and active that suppress osteoclast; And then treatment osteoporosis (McClung MR.et al.N Engl J Med, 2006,354; 821), this medicine is considered to become the potentiality of cookle medicine.Compare with the micromolecular compound of synthetic; Natural product has inborn meliority aspect the one-tenth property of medicine; Therefore from natural product, particularly start with from the effective constituent of high security natural product; Seeking novel mother nucleus structure and carry out structure activity study, is the important channel of finding into good, the safe osteosporosis resistant medicament of the property of medicine.In recent years, some natural triterpene compounds are in the news and have the effect that suppresses the osteoclast differentiation, can suppress differentiation, the maturation (Qiu S.X.et al.Chem Biol, 2007,14,860) of RANKL inductive osteoclast like the glucosides ACCX of Root of Roughleaf Ironweed alcohol; Pentacyclic triterpene boswellic acid derivatives AKBA can suppress differentiation, the maturation (Takada Y.et al.J Immun, 2006,176,3127) of osteoclast equally.Betulinic acid (Betulinic acid) and verivate thereof in antitumor, anti-AIDS, anti-inflammatory (Takada Y.et al.J Immun, 2003,171,3278; Saleem M.et al.Oncogene, 2004,23; 5203) etc. the aspect has good biological activity; Have certain promotion osteoblast differentiation active function (Lo Y.-C.et al, J.Agric.Food Chem.2010,58 simultaneously; 6643) also be that China is among the people with one of staple in the herbal medicine Williams Elder Twig (CN200410051613.4).
Summary of the invention
The objective of the invention is through betulonic acid is carried out composition optimizes, obtain active stronger candidate compound, lay the foundation, for the research of osteoporosis medicine provides condition for this compounds gets into clinical study.
Betulonic acid verivate provided by the invention has following general structure:
In the formula, Y represents CH
3Or NH
2
When the Y substituting group is CH
3The time, corresponding betulonic acid verivate is represented with the code name XJ-510 of design voluntarily; Y=NH
2The time, corresponding betulonic acid verivate is XJ-511;
Another object of the present invention is to provide the preparation method of this analog derivative.Above-claimed cpd I can make through following steps:
According to above-mentioned reaction formula, adopt ether solvent, mainly be in anhydrous diethyl ether, betulonic acid and dibrominated Mai Erduomu acid stirring at room to be reacted 4 hours, purification process gets compound i ii, yield 82%; Compound i ii reacted 12 hours with thioacetamide or thiocarbamide respectively in anhydrous ethanol solvent, 78 ℃ of temperature of reaction, and purifying gets the betulonic acid derivative I, comprises XJ-510 and XJ-511, yield 53%～62%.
A further object of the invention is the application of such betulonic acid verivate in the treatment medicine for treating osteoporosis; Through differentiation suppresses screening active ingredients to osteoclast, find that such betulonic acid verivate that aforesaid method obtains has the activity of remarkable inhibition osteoclast differentiation.This analog derivative suppresses the active IC of osteoclast differentiation
50＜5 μ M are higher than pentacyclic triterpene natural product Betulinic acid (IC
50=25 μ M).
The invention has the advantages that through the natural product betulonic acid is carried out structure of modification; At C2; Introduce and heterocycle the C3 position, has efficiently synthesized novel betulonic acid verivate, simple synthetic method; And, significantly strengthened this compounds and suppressed the activity that osteoclast breaks up through the structure of modification at this position.
Embodiment
Embodiment 1: the preparation of betulonic acid verivate XJ-510
0.45g betulonic acid and 0.18g dibrominated Mai Erduomu acid add in the 10mL anhydrous diethyl ether; Stirring at room 4 hours, reaction solution is used the 10ml saturated sodium bicarbonate solution respectively, the water washing of 10ml saturated common salt after adding the 40ml ether; Organic layer is used anhydrous sodium sulfate drying; (sherwood oil: purifying ETHYLE ACETATE=5: 1) gets 0.43g compound i v, productive rate 82% to thick product with silica gel column chromatography after boiling off organic solvent.
0.30g compound i v and 0.06g thioacetamide are joined in the 15mL absolute ethyl alcohol, back flow reaction 12 hours, reaction solution concentrates the back and adds 50mL water; ETHYLE ACETATE (100mL * 3) extraction; Merge organic layer, saturated aqueous common salt 30ml washing, boil off organic solvent after thick product with silica gel column chromatography (sherwood oil: purifying ETHYLE ACETATE=5: 1); Get 0.15g betulonic acid verivate XJ-510, productive rate 53%;
1H-NMR (CDCl
3): 4.67 (1H, s), 4.54 (1H, s), 2.98 (1H, m), 2.63 (1H, d J=15.4Hz), 2.57 (3H, s), 2.24 (4H, m); ESI-HRMS (m/z) [M+H]
+=510.3428.
Embodiment 2: the preparation of betulonic acid verivate XJ-511
The method that employing is similar to embodiment 1 prepares XJ-511, and difference is to substitute thioacetamide with thiocarbamide, gets betulonic acid verivate XJ-511, productive rate 62%.
1H-NMR(DMSO-d
6)：δ12.06(1H，br?s)，6.58(2H，br?s)，4.76(1H，s)，4.62(1H，s，)，2.96(1H，m)，2.46(1H，d，J＝18Hz)；ESI-HRMS(m/z)[M+H+]
+＝511.3363。
The used betulonic acid of embodiment and other reagent are commercially available analytical pure.
Embodiment 3: the betulonic acid verivate suppresses broken bone precursor cell differentiation test
Principle: the broken bone precursor cell of monokaryon carries out statistical study in noble cells factor mouse spinal fixation, embedding, section, dyeing.With contrast sham-operation mouse spinal fixation, embedding, section, dyeing, carry out statistical study.With contrast sham-operation mouse spinal fixation, embedding, section, dyeing, carry out statistical study.RANKL induces down with the contrast sham-operation, can merge gradually to be divided into the multinuclear mature osteoclast.Broken bone precursor cell does not possess the ability of dissolving sclerotin, has only the osteoclast of differentiation and maturation just to have the ability of dissolving sclerotin, and therefore, the level of differentiation of osteoclast can be reacted its molten bone ability.
Method: will break the bone precursor cell is that RAW264.7 is inoculated in 96 well culture plates 3000 cells/well.After treating that cell attachment spends the night, control group (adding 50nglmlRANKL) and experimental group (adding 50ng/mlRANKL and 5-20 μ M dosage betulonic acid verivate) group is set, changes liquid every other day, cultivated 3-5 days.After cellular control unit fusion fully; Distillation with 37 ℃ of preheatings is washed cell once; Added acetone fixed 30 seconds; The distillation washing cell of 37 ℃ of preheatings three times carries out TRAP dyeing then, obtains osteoclast at microscopically counting TRAP male multi-nucleus cell number (examining unnecessary 3) and forms percentage ratio.At first select 5 μ M, 10 μ M, the betulonic acid verivate of 20 μ M concentration suppresses active testing; Do not show obvious inhibiting activity like compound in 20 μ M concentration; Then do not do further analysis, still show better inhibited activity in 5 μ M concentration, then further test 2.5 μ M like compound; 1 μ M, the inhibition of 0.5 μ M and 0.1 μ M is active.
Test result: The selection result is the percent inhibition of when compound is various concentration, osteoclast being broken up.The betulonic acid verivate suppresses the test result of osteoclast precursor RAW264.7 differentiation, sees table 1:
Table 1
aPercent inhibition=100%-osteoclast forms percentage ratio
Table 1 result shows that this compounds still shows very high inhibition activity in 5 μ M concentration.
In sum; Such betulonic acid verivate is as the osteoclast differentiation inhibitors; Prevent, delay or treat osteoporosis in preparation by the osteoclast mediation; Rheumatic arthritis, rheumatoid arthritis has important use in the relative disease medicines such as osteoclasia that periodontitis and cancer bone metastasis cause.
Claims (3)
1. one kind is suppressed the betulonic acid verivate that osteoclast breaks up, and it is characterized in that this betulonic acid verivate has following structure:
In the formula, Y represents CH
3Or NH
2
2. a betulonic acid thereof, derivatives thereof for preparing claim 1 is characterized in that, carries out according to following operation steps:
According to above-mentioned reaction formula, earlier betulonic acid iii and dibrominated Mai Erduomu acid are added in the anhydrous diethyl ether, stirring at room 4 hours, purifying gets compound i v; Again compound i v and thioacetamide or thiocarbamide are joined in the absolute ethyl alcohol, back flow reaction 12 hours, reaction solution concentrates the back extraction; Merge organic layer; The saturated common salt water washing boils off behind the organic solvent thick product and use purification by silica gel column chromatography, must the betulonic acid derivative I.
3. the application method of the betulonic acid verivate of a claim 1 is characterized in that, the application of this betulonic acid derivative I in preparation treatment medicine for treating osteoporosis.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1682740A (en) * | 2005-03-11 | 2005-10-19 | 中国药科大学 | Use of pentacylic triterpene compounds in preparing glycogenic phosphorylase inhibitor |
| WO2007101873A2 (en) * | 2006-03-07 | 2007-09-13 | Sndv Sprl | Betulonic acid esters and betulinic acid polyalkylene-glycol derivatives for the treatment of viral infection and cancer |
| WO2008063318A2 (en) * | 2006-10-12 | 2008-05-29 | The Texas A & M University System And Safe Et Al | Betulinic acid, derivatives and analogs thereof and uses therefor |
| CN101412743A (en) * | 2008-11-27 | 2009-04-22 | 中国药科大学 | 2 alpha-hydroxy-substituted white birch keto acid, preparation thereof and anti-tumor use |
| CN101619088A (en) * | 2009-08-04 | 2010-01-06 | 上海朴颐生物科技有限公司 | Maslinic acid derivative as well as preparation and application thereof |
-
2011
- 2011-03-11 CN CN2011100596092A patent/CN102167720B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1682740A (en) * | 2005-03-11 | 2005-10-19 | 中国药科大学 | Use of pentacylic triterpene compounds in preparing glycogenic phosphorylase inhibitor |
| WO2007101873A2 (en) * | 2006-03-07 | 2007-09-13 | Sndv Sprl | Betulonic acid esters and betulinic acid polyalkylene-glycol derivatives for the treatment of viral infection and cancer |
| WO2008063318A2 (en) * | 2006-10-12 | 2008-05-29 | The Texas A & M University System And Safe Et Al | Betulinic acid, derivatives and analogs thereof and uses therefor |
| CN101412743A (en) * | 2008-11-27 | 2009-04-22 | 中国药科大学 | 2 alpha-hydroxy-substituted white birch keto acid, preparation thereof and anti-tumor use |
| CN101619088A (en) * | 2009-08-04 | 2010-01-06 | 上海朴颐生物科技有限公司 | Maslinic acid derivative as well as preparation and application thereof |
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