CN106749406B - A kind of 2- alkyl substituted imidazole base double phosphinic acid compounds and preparation method thereof, application - Google Patents

A kind of 2- alkyl substituted imidazole base double phosphinic acid compounds and preparation method thereof, application Download PDF

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CN106749406B
CN106749406B CN201611253873.9A CN201611253873A CN106749406B CN 106749406 B CN106749406 B CN 106749406B CN 201611253873 A CN201611253873 A CN 201611253873A CN 106749406 B CN106749406 B CN 106749406B
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osteoclast
substituted imidazole
alkyl substituted
cell
phosphinic acid
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CN106749406A (en
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邱玲
林建国
吕高超
李珂
彭莹
罗世能
王珊珊
赵学玉
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Jiangsu Institute of Nuclear Medicine
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    • C07FACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02Phosphorus compounds
    • C07F9/547Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
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    • C07F9/6506Five-membered rings having the nitrogen atoms in positions 1 and 3

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Abstract

The invention belongs to field of medicinal chemistry, more particularly to a kind of 2- alkyl substituted imidazole base double phosphinic acid compounds and the preparation method and application thereof, the position the 2- alkyl substituted imidazole base double phosphinic acid compounds are larger to the non-toxic concn section of osteoclast precursor, the formation of osteoclast can be significantly inhibited simultaneously, actin ring can farthest be destroyed, and regulate and control the expression of related gene, by reducing MMP-9 in osteoclast, Cathepsin K, c-Fos, NFATcl, the mRNA level in-site expression of the osteoclast formations Research of predicting markers gene such as RANK increases to inhibit osteoclast formation, and inhibit the bone resorption function of osteoclast by reducing the expression of TRPV5 and integrin β 3 caused by RANKL raising, it can be used as osteoclast suppression Preparation, solves that diphosphonate in the prior art is low to osteoclast inhibitory effect, and toxicity is big, and the problem that side effect is big.

Description

A kind of 2- alkyl substituted imidazole base double phosphinic acid compounds and preparation method thereof, application
Technical field
The invention belongs to field of medicinal chemistry, and in particular to a kind of 2- alkyl substituted imidazole base double phosphinic acid compounds and its Preparation method, application.
Background technique
Diphosphonate (Bisphosphonates, BPs) is the kind new medicine for all kinds of bone disorders and calcium metabolism disease Object can inhibit osteoclast activity, to inhibit bone absorption, clinically extensively specifically in conjunction with the hydroxyapatite in sclerotin It is general to be used to treat osteoporosis, scleromalacia, hypercalcemia illness and tumour related bone conditions etc..
Pluripotential hemopoietic stem cell of the osteoclast (osteoclast, OC) in marrow.Pluripotential hemopoietic stem cell is first It is first divided into macrophage system clonogenic unit, as osteoclast precursor under the action of the factors such as M-CSF, further exists later Fusion forms multicore and the osteoclast with bone resorption function under the stimulation of the factors such as RANKL.RAW264.7 cell is mouse The osteoclast precursor of source property, from Abelson mouse leukemia virus induction BALB/c lead to mice developing tumor after receive The cell strain that collection mouse ascites mononuclear macrophage obtains, is considered representing the osteoclast precursor cells in early differentiation stage.
" separation of bisphosphonate class of drugs reversed-phase liquid chromatography and application study " (Xie Zan, medical and health science and technology volume, 20061215) bisphosphonate class of drugs such as zoledronic acid (ZL) are disclosed with powerful anti-bone resorption and its mechanism, are passed through It is directly changed the form of osteoclast, actin is inhibited to be formed, interferes absorption and reabsorption of the osteoclast to bone;Also inhibit The vicious circle for being referred to as " seed and soil mechanism " between tumour and bon e formation, suppression are broken in the release of peptides in bone matrix Growth of tumour cell processed.However, people are about zoledronic acid to osteoclastic thin at present due to the complexity of zoledronic acid mechanism of action The mechanism of action that born of the same parents inhibit is still unclear that scientific research personnel is limited for the improvement of existing diphosphonate, so that existing double Effect of the phosphonate in terms for the treatment of metabolic bone disease is lower, and toxicity is big, and along with very big side effect.How to existing Zoledronic acid (ZL) molecule improves the inhibition improved to understand mechanism of drug action to the maximum extent to osteoclast and makees With, and reduce side effect, it has also become this field big technical problem urgently to be resolved.
Summary of the invention
The technical problem to be solved in the present invention is that diphosphonate in the prior art is low to osteoclast inhibitory effect, poison Property is big, and side effect is big, to provide, a kind of pair of osteoclast inhibitory effect be excellent, nontoxic and Small side effects medicines structure.
For this purpose, having and being tied shown in formula (I) the present invention provides a kind of 2- alkyl substituted imidazole base double phosphinic acid compounds Structure,
Wherein, R1 is the alkyl of C1-C10;C1-C10 halogenated alkyl;C1-C10 alkoxy;Or the C1-C10 that halogen replaces Alkoxy.
Optionally, the position 2- alkyl substituted imidazole base double phosphinic acid compounds, the R1 are the alkyl of C1-C6.
Optionally, the position 2- alkyl substituted imidazole base double phosphinic acid compounds, the R1 are the alkyl of C1-C4.
Optionally, the position 2- alkyl substituted imidazole base double phosphinic acid compounds, the alkyl of the C1-C4 are methyl, second Base, propyl, isopropyl, normal-butyl, sec-butyl, tert-butyl or isobutyl group.
The present invention provides a kind of officinal salt of above compound, hydrate, solvate, unformed body, monocrystalline and Eutectic.
The position the 2- alkyl substituted imidazole base double phosphinic acid compounds are prepared the present invention provides a kind of, including are walked as follows It is rapid:
(1) 2- shown in formula A-I alkyl substituted imidazole based compound, alkali and tetrabutylammonium bromide are dissolved in organic solvent In, mixing is stirred at room temperature, bromoacetate is then slowly added dropwise thereto, then the back flow reaction 6-10h at 30-50 DEG C, with By being filtered, washed and dried, formula A-II compound represented is obtained;
(2) formula A-II compound represented is soluble in water, after addition acid solution tune pH value is 1-2, in 80-120 DEG C Lower back flow reaction 5-10h, resulting reaction solution is spin-dried for, and acetone is added thereto and obtains white solid, then filters, recrystallizes, Obtain formula A-III compound represented;
(3) phosphoric acid is added into formula A-III compound represented and polyethylene glycol reacts 0.2-1h at 80-120 DEG C, so After gained reaction solution is cooled to 60-70 DEG C afterwards, phosphorus trichloride is slowly added dropwise in Xiang Suoshu reaction solution, it is then anti-in 80-120 DEG C 1-5h is answered, concentrated hydrochloric acid is then added, 80-120 DEG C of reflux 3-8h is continued at, pours into temperature after gained reaction solution is cooled to room temperature In 0-6 DEG C of ethyl alcohol, to there is white solid precipitation, filters, be dried in vacuo to get formula A-IV compound represented, as institute is arrived The position the 2- alkyl substituted imidazole base double phosphinic acid compounds stated;
Preferably, the preparation method of the position 2- alkyl substituted imidazole base double phosphinic acid compounds, includes the following steps:
(1) 2- shown in formula A-I alkyl substituted imidazole based compound, alkali and tetrabutylammonium bromide are dissolved in organic solvent In, mixing is stirred at room temperature, bromoacetate is then slowly added dropwise thereto, then the back flow reaction 7-9h at 35-45 DEG C, then It is filtered, washed and dried drying, obtains formula A-II compound represented;
(2) formula A-II compound represented is soluble in water, after addition acid solution tune pH value is 1-2, in 90-110 DEG C Lower back flow reaction 6-8h, resulting reaction solution is spin-dried for, and acetone is added thereto and obtains white solid, then filters, recrystallizes, obtains To formula A-III compound represented;
(3) phosphoric acid is added into formula A-III compound represented and polyethylene glycol reacts 0.4-0.6h at 90-110 DEG C, Then after gained reaction solution being cooled to 63-67 DEG C, phosphorus trichloride is slowly added dropwise in Xiang Suoshu reaction solution, then in 90-110 DEG C 2-4h is reacted, concentrated hydrochloric acid is then added, continues at 90-110 DEG C of reflux 4-6h, pours into temperature after gained reaction solution is cooled to room temperature Degree filters, vacuum drying is to get formula A-IV compound represented is arrived, as to have white solid precipitation in 0-4 DEG C of ethyl alcohol The position the 2- alkyl substituted imidazole base double phosphinic acid compounds.
Preferably, the alkali is selected from KOH, NaOH, K2CO3、Na2CO3, sodium tert-butoxide, at least one of sodium ethoxide.
Preferably, the alkali is KOH and K2CO3
The organic solvent is polar aprotic solvent.
Preferably, the organic solvent is methylene chloride, chloroform and/or tetrahydrofuran.
Preferably, on the basis of the mole of the 2- alkyl substituted imidazole based compounds shown in the formula A-I, the uncle Sodium butoxide and the respective dosage of sodium ethoxide are 1.1-1.5 moles times and measure that the dosage of the tetrabutylammonium bromide is rubbed for 0.03-0.07 You measure again, and the dosage of the bromoacetate is 0.8-1.2 moles times and measures.
Preferably, in the step (1), with rubbing for 2- alkyl substituted imidazole based compounds shown in the formula A-I On the basis of your amount, the dosage of the KOH is 1.3-1.7 moles times and measures, the K2CO3Dosage be 0.8-1.2 moles times and measure, institute The dosage for stating tetrabutylammonium bromide is 0.03-0.07 moles times and measures that the dosage of the bromoacetate is 0.8-1.2 moles times Amount.
Preferably, in the step (1), with rubbing for 2- alkyl substituted imidazole based compounds shown in the formula A-I On the basis of your amount, the dosage of the KOH is 1.5 moles times and measures, the K2CO3Dosage be 1 mole times and measure, the tetrabutyl bromine The dosage for changing ammonium is 0.05 mole times and measures that the dosage of the bromo acetoacetic ester is 1 mole times and measures.
Preferably, in the step (3), on the basis of the mole of the formula A-III compound represented, the phosphorus The dosage of acid is 0.8-1.2 moles times and measures that the dosage of the polyethylene glycol is 1.3-1.7 moles times and measures, the phosphorus trichloride Dosage is 0.8-1.2 moles times and measures.
Preferably, in the step (3), on the basis of the mole of the formula A-III compound represented, the phosphorus The dosage of acid is 1 mole times and measures that the dosage of the polyethylene glycol is 1.5 moles times and measures that the dosage of the phosphorus trichloride is 1 mole It measures again.
The present invention provides a kind of the above-mentioned position 2- alkyl substituted imidazole base double phosphinic acid compounds or above-mentioned method systems The standby position 2- alkyl substituted imidazole base double phosphinic acid compounds are preparing the purposes in osteoclast inhibitors.
The present invention provides a kind of osteoclast inhibitors, with the above-mentioned position 2- alkyl substituted imidazole base double phosphinic acid compounds Or the position the 2- alkyl substituted imidazole base double phosphinic acid compounds of above-mentioned method preparation are effective component.
Optionally, using the position the 2- alkyl substituted imidazole base double phosphinic acid compounds as effective component, according to common process, Selectively add clinically-acceptable preparation made of customary adjuvant.
Technical solution of the present invention has the advantages that
1. 2- alkyl substituted imidazole base double phosphinic acid compounds of the present invention are to the non-toxic concn of osteoclast precursor Section is larger, and is in concentration dependent, and the formation of osteoclast can be significantly inhibited under lower concentration, can be farthest Actin ring is destroyed, or even causes the death of osteoclast, and regulate and control the expression of related gene, is caused by reducing RANKL Osteoclast in the osteoclast formations Research of predicting markers gene such as MMP-9, Cathepsin K, c-Fos, NFATc1, RANK MRNA level in-site expression increases to inhibit osteoclast formation, and by reducing by 3 table of TRPV5 and integrin β caused by RANKL The bone resorption function of inhibiting osteoclast up to increasing has significant inhibiting effect for osteoclast, can be used as osteoclastic Cytostatics.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 a is control group RAW264.7 cell;
Fig. 1 b be RANKL induction RAW264.7 cell for 24 hours when state;
State when Fig. 1 c is RANKL induction RAW264.7 cell 48h;
State when Fig. 1 d is RANKL induction RAW264.7 cell 72h;
State when Fig. 1 e is RANKL induction RAW264.7 cell 96h;
Fig. 2 a- (1) is control group RAW264.7 cell;
Fig. 2 a- (2) is that the huge TRAP positive cell of a large amount of multicores is formed after RANKL is induced;
Fig. 2 b- (1) is control group RAW264.7 cell;
Fig. 2 b- (2) is the actin ring fluorescent staining figure that a large amount of multicore megabacteriums are formed after RANKL is induced;
Fig. 3 is inhibited proliferation (72h) of the BPs to RAW264.7 cell;
Fig. 4 a is non-toxic concn of the ZL to RAW264.7;
Fig. 4 b is non-toxic concn of the MIDP to RAW264.7;
Fig. 4 c is non-toxic concn of the EIDP to RAW264.7;
Fig. 4 d is non-toxic concn of the PIDP to RAW264.7;
Fig. 4 e is non-toxic concn of the BIDP to RAW264.7;
Fig. 5 a is influence of the PIDP in non-toxic concn section to the RANKL osteoclast formation induced;
Fig. 5 b is influence of the BIDP in non-toxic concn section to the RANKL osteoclast formation induced;
Fig. 5 c is influence of the ZL in non-toxic concn section to the RANKL osteoclast formation induced;
Fig. 5 d is influence of the MIDP in non-toxic concn section to the RANKL osteoclast formation induced;
Fig. 5 e is influence of the EIDP in non-toxic concn section to the RANKL osteoclast formation induced;
Fig. 6 is the active influence of osteoclast TRAP that non-toxic concn PIDP induces RANKL;
Fig. 7 is influence of the non-toxic concn PIDP to the RANKL osteoclast actin ring structure induced;
Fig. 8 a is after 0 μM of PIDP and 0ng/mlRANKL effect, to the TRAP colored graph of RAW264.7;
Fig. 8 b is after 0 μM of PIDP and 50ng/mlRANKL effect, to the TRAP colored graph of RAW264.7;
Fig. 8 c is after 3.125 μM of PIDP and 50ng/mlRANKL effect, to the TRAP colored graph of RAW264.7;
Fig. 8 d is after 6.25 μM of PIDP and 50ng/mlRANKL effect, to the TRAP colored graph of RAW264.7;
Fig. 8 e is after 12.5 μM of PIDP and 50ng/mlRANKL effect, to the TRAP colored graph of RAW264.7;
Fig. 9 is PIDP to osteoclast formation related gene MMP-9, Cathepsin K, c-Fos, NFATc1, RANK The influence of mRNA level in-site expression;
Figure 10 is the influence of mRNA level in-site expression of the PIDP to bone resorption related gene TRPV5, integrin β 3;
Figure 11 is the influence that PIDP expresses osteoclast formation GAP-associated protein GAP c-Fos, NFATc1;
Figure 12 is influence of the PIDP to osteoclast formation associated signal paths.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention. In addition, technical characteristic involved in invention described below different embodiments is as long as they do not conflict with each other It can be combined with each other.
The reagent being related in following embodiments is as follows:
2-methylimidazole, 2- ethyl imidazol(e), 2- propyl imidazole, 2 isopropyl imidazole, 2- butyl imidazole, 2- sec-butyl miaow Azoles, 2- isobutyl group imidazoles, 2- t-butyl imidazole, bromoacetate, phosphoric acid, phosphorus trichloride, potassium hydroxide, potassium carbonate, concentrated hydrochloric acid, Anhydrous sodium sulfate, polyethylene glycol, methylene chloride, ethyl alcohol, acetone.All reagents are that analysis is pure, using preceding not purified, are purchased In Aladdin Reagent Company.
The manufacturer of zoledronic acid is Aladdin.
The instrument being related in following embodiments is as follows:
Nuclear Magnetic Resonance (Bruker DRX-500), electrospray mass spectrometer (Waters ZMD4000), elemental analyser (Elementar Vario EL III)
Embodiment 1
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At, wherein R1 is methyl, there is formula (II) structure:
The synthetic route of compound ii are as follows:
(1) prepared by compound ii intermediate 1
By 2-methylimidazole 8.2g (0.1mol), KOH 8.4g (0.15mol), K2CO313.8g (0.1mol) and the tetrabutyl Ammonium bromide 1.6g (0.005mol) is dissolved in 75mL methylene chloride, and after 0.5h is stirred at room temperature, bromoacetate is slowly added dropwise 0.1mol (11.2mL), is added dropwise, and flow back 8h at 39 DEG C, filtering, and three times, anhydrous sodium sulfate is dry for saturated common salt water washing filtrate Dry, 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtain grease, i.e., the described compound ii intermediate 1;
(2) prepared by compound ii intermediate 2
Taking II intermediate 1 of above compound is that 8.4g (0.05mol) is dissolved in 100mL water, and concentrated hydrochloric acid adjusts solution ph Flow back 7h at 1.5,100 DEG C, after reaction, 25 DEG C of gained reaction solution is directly evaporated under reduced pressure to drop and is no longer oozed, is added Acetone 300mL obtains white solid, and filtering, recrystallisation from isopropanol obtains white crystal to get the compound ii intermediate 2;
(3) prepared by compound ii
Taking II intermediate 2 of above compound is 3.5g (0.025mol), H3PO42.45g (0.025mol) and polyethylene glycol 400, to react 0.5h at 100 DEG C of 15g (0.037mol), after being cooled to 65 DEG C, are slowly added dropwise phosphorus trichloride 0.025mol, are added dropwise It finishes, continues 100 DEG C of reaction 3h, 30mL concentrated hydrochloric acid is then added, flow back 5h, and reaction terminates, and is cooled to room temperature, by gained reaction solution It pours into 2 DEG C of ethyl alcohol, there is white solid precipitation, filter, be dried in vacuo to get the compound ii.
1H-NMR(500MHz,D2O)7.38(s,1H,IMZ-H),7.13(s,1H,IMZ-H),4.50(s,2H,N–CH2), 2.51(s,3H,CH3) .ESI-MS, m/z (%): 285 (100)=M-H+, Anal.calcd for C5H10N2O7P2(%): C, 22.07;H,3.70;N,10.30;Found (%): C, 22.27;H,3.78;N,10.24.
Embodiment 2
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At, wherein R1 is ethyl, there is formula (III) structure:
The synthetic route of compound III are as follows:
(1) prepared by compound III intermediate 1
Take 2- ethyl imidazol(e) 9.6g (0.1mol), KOH 8.4g (0.15mol), K2CO313.8g (0.1mol) and the tetrabutyl Ammonium bromide 1.6g (0.005mol) is dissolved in 75mL methylene chloride, and after 0.5h is stirred at room temperature, bromoacetate is slowly added dropwise 0.1mol (11.2mL), is added dropwise, and flow back 8h at 39 DEG C, filtering, and three times, anhydrous sodium sulfate is dry for saturated common salt water washing filtrate Dry, 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtain grease, i.e., the described compound III intermediate 1;
(2) prepared by compound III intermediate 2
Taking III intermediate 1 of above compound is that 9.1g (0.05mol) is dissolved in 100mL water, enriching hydrochloric acid conditioning solution pH Flow back 7h at 1.0,100 DEG C of value.After reaction, 25 DEG C of gained reaction solution are directly evaporated under reduced pressure to drop and are no longer oozed, added Enter acetone 300mL, obtain white solid, filter, recrystallisation from isopropanol obtains white crystal, i.e., the described compound III intermediate 2;
(3) prepared by compound III
Taking III intermediate 2 of above compound is 3.85g (0.025mol), H3PO42.45g (0.025mol) and polyethylene glycol 400, to react 0.5h at 100 DEG C of 15g (0.037mol), after being cooled to 65 DEG C, are slowly added dropwise phosphorus trichloride 0.025mol (2.2ml), is added dropwise, and continues 100 DEG C of reaction 3h, and 30mL concentrated hydrochloric acid is then added, and flow back 5h, and reaction terminates, and is cooled to room temperature, Reaction solution is poured into 5 DEG C of ethyl alcohol, there is white solid precipitation, filtered, vacuum drying obtains the compound III.
1H-NMR(500MHz,D2O)7.38(s,1H,IMZ-H),7.13(s,1H,IMZ-H),4.50(s,2H,N–CH2), 2.99(m,2H,CH2),1.28(t,3H,CH3) .ESI-MS, m/z (%): 299=[M-H+], Anal.Calculated: C7H14N2O7P2(%): C, 28.01;H,4.70;N,9.33;Found (%): C, 29.12;H,4.82;N,9.51.
Embodiment 3
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At, wherein R1 is propyl, there is formula (IV) structure:
The synthetic route of compounds Ⅳ are as follows:
(1) prepared by compounds Ⅳ intermediate 1
Take 2- propyl imidazole 11.0g (0.1mol), KOH 8.4g (0.15mol), K2CO313.8g (0.1mol) He Siding Base ammonium bromide 1.6g (0.005mol) is dissolved in 75mL methylene chloride, and after 0.5h is stirred at room temperature, bromoacetate is slowly added dropwise 0.1mol (11.2mL), is added dropwise, and flow back 9h at 39 DEG C, filtering, and three times, anhydrous sodium sulfate is dry for saturated common salt water washing filtrate Dry, 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtain grease to get the compounds Ⅳ intermediate 1;
(2) prepared by compounds Ⅳ intermediate 2
Taking IV intermediate 1 of above compound is that 9.8g (0.05mol) is dissolved in 100mL water, enriching hydrochloric acid conditioning solution pH Flow back 6h at 2,110 DEG C of value, after reaction, 25 DEG C of gained reaction solution is directly evaporated under reduced pressure to drop and is no longer oozed, is added Acetone 300mL, obtains white solid.Filtering, recrystallisation from isopropanol obtain white crystal, i.e., the described compounds Ⅳ intermediate 2;
(3) prepared by compounds Ⅳ
Taking IV intermediate 2 of above compound is 4.2g (0.025mol), H3PO42.45g (0.025mol) and polyethylene glycol 400, to react 0.4h at 110 DEG C of 15g (0.037mol), after being cooled to 67 DEG C, are slowly added dropwise phosphorus trichloride 0.025mol (2.2ml), is added dropwise, and continues 110 DEG C of reaction 2h, and 30mL concentrated hydrochloric acid is then added, and flow back 6h, and reaction terminates, and is cooled to room temperature, Gained reaction solution is poured into 5 DEG C of ethyl alcohol, there is white solid precipitation, filtered, vacuum drying obtains the compounds Ⅳ.
1H-NMR (500MHz, D2O) 7.38 (s, 1H, IMZ-H), 7.13 (s, 1H, IMZ-H), 4.50 (s, 2H, N-CH2), 2.99(m,2H,CH2),1.61(m,2H,CH2),0.83(m,3H,CH3) .ESI-MS, m/z (%): 313=[M-H+], Anal.calculated:C8H16N2O7P2(%): C, 30.58;H,5.13;N,8.92;Found (%): C, 31.62;H,5.32; N,9.05.
Embodiment 4
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At, wherein R1 is normal-butyl, there is formula (V) structure:
The synthetic route of compound V are as follows:
(1) prepared by V intermediate 1 of compound
Take 2- butyl imidazole 12.4g (0.1mol), KOH 8.4g (0.15mol), K2CO313.8g (0.1mol) He Siding Base ammonium bromide 1.6g (0.005mol) is dissolved in 75mL methylene chloride, and after 0.5h is stirred at room temperature, bromoacetate is slowly added dropwise 0.1mol (11.2mL), is added dropwise, and flow back 9h at 35 DEG C.Filtering, three times, anhydrous sodium sulfate is dry for saturated common salt water washing filtrate Dry, 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtain grease, i.e., described V intermediate 1 of compound;
(2) prepared by V intermediate 2 of compound
Taking V intermediate 1 of above compound is that 10.5g (0.05mol) is dissolved in 100mL water, enriching hydrochloric acid conditioning solution pH Flow back 8h at 2,110 DEG C of value, after reaction, 25 DEG C of gained reaction solution is directly evaporated under reduced pressure to drop and is no longer oozed, is added Acetone 300mL, obtains white solid.Filtering, recrystallisation from isopropanol obtain white crystal, i.e., described V intermediate 2 of compound;
(3) prepared by compound V
Taking V intermediate 2 of above compound is 4.6g (0.025mol), H3PO42.45g (0.025mol) and polyethylene glycol 400, to react 0.6h at 100 DEG C of 15g (0.037mol), after being cooled to 65 DEG C, are slowly added dropwise phosphorus trichloride 0.025mol (2.2ml), is added dropwise, and continues 110 DEG C of reaction 4h, and 30mL concentrated hydrochloric acid is then added, and flow back 8h, and reaction terminates, and is cooled to room temperature, Gained reaction solution is poured into 3 DEG C of ethyl alcohol, there is white solid precipitation, filtered, vacuum drying obtains the compound V.
1H-NMR(500MHz,D2O):7.38(s,1H,IMZ-H),7.13(s,1H,IMZ-H),4.50(s,2H,N–CH2), 2.99(m,2H,CH2),1.61(m,2H,CH2),1.27(m,2H,CH2),0.8(m,3H,CH3) .ESI-MS, m/z (%): 327 =[M-H+], Anal.Calc.for C9H18N2O7P2(%): C 32.94;H,5.53;N, 8.54.Found (%): C, 33.12; H,3.69;N,8.31.
Embodiment 5
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At, wherein R1 is isopropyl, there is formula (VI) structure:
The synthetic route of compound VI are as follows:
(1) prepared by VI intermediate 1 of compound
Take 2 isopropyl imidazole 11.0g (0.1mol), KOH 7.3g (0.13mol), K2CO316.6g (0.12mol) and four Butylammonium bromide 0.97g (3mmol) is dissolved in 75mL chloroform, and after 0.5h is stirred at room temperature, bromoacetate 0.08mol is slowly added dropwise (8.9ml), is added dropwise, and flow back 6h at 30 DEG C, filtering, and three times, anhydrous sodium sulfate is dry for saturated common salt water washing filtrate, and 25 DEG C Vacuum distillation organic phase is no longer oozed to drop, obtains grease, i.e., described VI intermediate 1 of compound.
(2) prepared by VI intermediate 2 of compound
Taking VI intermediate 1 of above compound is that 9.8g (0.05mol) is dissolved in 100mL water, and concentrated hydrochloric acid adjusts solution ph Flow back 10h at 1.8,80 DEG C, after reaction, 25 DEG C of gained reaction solution is directly evaporated under reduced pressure to drop and is no longer oozed, is added Acetone 300mL obtains white solid, and filtering, recrystallisation from isopropanol obtains white crystal, i.e., described VI intermediate 2 of compound;
(3) prepared by compound VI
Taking VI intermediate 2 of above compound is 4.2g (0.025mol), H3PO42.0g (0.02mol) and polyethylene glycol 400 It is to react 0.2h at 80 DEG C of 13g (0.0325mol), after being cooled to 60 DEG C, is slowly added dropwise phosphorus trichloride 0.02mol (1.7mL), drips After adding, continue 80 DEG C of reaction 5h, 30mL concentrated hydrochloric acid is then added, flow back 3h, and reaction terminates, and is cooled to room temperature, and gained is anti- It answers liquid to pour into 1 DEG C of ethyl alcohol, there is white solid precipitation, filter, vacuum drying obtains the compound VI.
1H-NMR(500MHz,D2O):7.44(d,1H,CH-ring),6.82(d,1H,CH-ring),4.56(t,2H,N– CH2),3.55(m,1H,CH(CH3)2),1.25(d,6H,CH3) .ESI-MS, m/z (%): 313=[M-H+], Anal.calculated:C8H16N2O7P2(%): C, 30.58;H,5.13;N,8.92;Found (%): C, 31.62;H,5.32; N,9.05.
Embodiment 6
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At wherein R1For sec-butyl, there is formula (VII) structure:
The synthetic route of compound VII are as follows:
(1) prepared by VII intermediate 1 of compound
Take 2- sec-butyl imidazoles 12.4g (0.1mol), NaOH 6.8g (0.17mol), Na2CO38.5g (0.08mol) and Tetrabutylammonium bromide 2.3g (7mmol) is dissolved in 75mL tetrahydrofuran, and after 0.5h is stirred at room temperature, bromoacetate is slowly added dropwise 0.1mol (11.2mL), is added dropwise, and flow back 9h at 45 DEG C.Filtering, three times, anhydrous sodium sulfate is dry for saturated common salt water washing filtrate Dry, 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtain grease, i.e., described VII intermediate 1 of compound;
(2) prepared by VII intermediate 2 of compound
Taking VII intermediate 1 of above compound is that 10.5g (0.05mol) is dissolved in 100mL water, enriching hydrochloric acid conditioning solution pH Gained reaction solution is spin-dried for by the 8h that flows back at 1.2,90 DEG C of value after reaction, and 25 DEG C of reaction solution is directly evaporated under reduced pressure to drop not It oozes again, acetone 300mL is added, obtains white solid, filter, recrystallisation from isopropanol obtains white crystal, i.e., in the described compound VII Mesosome 2;
(3) prepared by compound VII
Taking VII intermediate 2 of above compound is 4.6g (0.025mol), H3PO42.9g (0.03mol) and polyethylene glycol 400 It is to react 0.8h at 90 DEG C of 17g (0.0425mol), after being cooled to 70 DEG C, is slowly added dropwise phosphorus trichloride 0.03mol (2.6mL), drips After adding, continue 120 DEG C of reaction 1h, 30mL concentrated hydrochloric acid is then added, flow back 3h, and reaction terminates, and is cooled to room temperature, by reaction solution It pours into 2 DEG C of ethyl alcohol, there is white solid precipitation, filter, vacuum drying obtains the compound VII.
1H-NMR(500MHz,D2O)7.38(s,1H,IMZ-H),7.13(s,1H,IMZ-H),4.50(s,2H,N–CH2), 2.51(m,2H,CH2),1.81(m,1H,CH),1.26(d,2H,CH3),0.91(t,3H,CH3) .ESI-MS, m/z (%): 327 =[M-H+],Anal.Calc.for C9H18N2O7P2(%): C 32.94, H 5.53, N 8.54.Found (%): C, 33.12; H,3.69;N,8.31.
Embodiment 7
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At wherein R1For tert-butyl, there is formula (VIII) structure:
The synthetic route of compound VIII are as follows:
(1) prepared by VIII intermediate 1 of compound
Take 2- t-butyl imidazole 12.4g (0.1mol), KOH 9.5g (0.17mol), K2CO311.1g (0.08mol) and four Butylammonium bromide 1.6g (5mmol) is dissolved in 75mL methylene chloride, and after 0.5h is stirred at room temperature, bromoacetate is slowly added dropwise 0.12mol (13.3mL), after being added dropwise to complete, flow back 10h at 50 DEG C.Filtering, saturated common salt water washing filtrate three times, anhydrous slufuric acid Sodium is dry, and 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtains grease, i.e., described VIII intermediate 1 of compound;
(2) prepared by VIII intermediate 2 of compound
Taking VI intermediate 1 of above compound is that 10.5g (0.05mol) is dissolved in 100mL water, and concentrated hydrochloric acid adjusts solution ph Flow back 6h at 1.5,110 DEG C, and after reaction, gained reaction solution is spin-dried for, and 25 DEG C of the reaction solution is directly evaporated under reduced pressure to liquid Drop no longer oozes, and acetone 300mL is added, obtains white solid, filters, and recrystallisation from isopropanol obtains white crystal, i.e., the described compound VIII intermediate 2;
(3) prepared by compound VIII
Taking VIII intermediate 2 of above compound is 4.6g (0.025mol), H3PO42.4g (0.025mol) and polyethylene glycol 400, to react 0.6h at 90 DEG C of 15g (0.037mol), after being cooled to 67 DEG C, are slowly added dropwise phosphorus trichloride 0.025mol (2.2mL) after being added dropwise, continues 90 DEG C of reaction 4h.Then the dense HCl of 30mL is added, continues the 4h that flows back, reaction terminates, is cooled to Room temperature pours into gained reaction solution in 0 DEG C of ethyl alcohol, there is white solid precipitation, filters, and vacuum drying obtains the compound VIII.
1H-NMR(500MHz,D2O)7.38(s,1H,IMZ-H),7.13(s,1H,IMZ-H),4.50(s,2H,N–CH2), 2.51(m,2H,CH2),1.81(m,1H,CH),0.91(d,6H,CH3) .ESI-MS, m/z (%): 327=[M-H+], Anal.Calc.for C9H18N2O7P2(%): C, 32.94;H,5.53;N, 8.54.Found (%): C 33.12;H 3.69;N 8.31.
Embodiment 8
The conjunction of the position 2- alkyl substituted imidazole base double phosphinic acid compounds with structure shown in formula (I) described in the present embodiment At wherein R1For isobutyl group, there is formula (Ⅸ) structure:
The synthetic route of compound Ⅸ are as follows:
(1) prepared by Ⅸ intermediate 1 of compound
Take 2- isobutyl group imidazoles 12.4g (0.1mol), sodium tert-butoxide 10.6g (0.11mol), sodium ethoxide 7.5g (0.11mol) and tetrabutylammonium bromide 1.6g (5mmol) are dissolved in 75mL methylene chloride, after 0.5h is stirred at room temperature, are slowly added dropwise Bromoacetate 0.1mol (11.2mL), is added dropwise, and flow back 8h at 39 DEG C.Filtering, saturated common salt water washing filtrate three times, nothing Aqueous sodium persulfate is dry, and 25 DEG C of vacuum distillation organic phases are no longer oozed to drop, obtains grease, i.e. Ⅸ intermediate 1 of compound.
(2) prepared by Ⅸ intermediate 2 of compound
Taking Ⅸ intermediate 1 of above compound is that 10.5g (0.05mol) is dissolved in 100mL water, enriching hydrochloric acid conditioning solution pH Flow back 5h at 1.5,120 DEG C of value, and after reaction, reaction solution is spin-dried for, and 25 DEG C of gained reaction solution are directly evaporated under reduced pressure to drop It no longer oozes, acetone 300mL is added, obtains white solid, filter, recrystallisation from isopropanol obtains white crystal, i.e., the described compound Ⅸ Intermediate 2.
(3) prepared by compound Ⅸ
Taking Ⅸ intermediate 2 of above compound is 4.6g (0.025mol), H3PO42.4g (0.025mol) and polyethylene glycol 400 is react 1h at 110 DEG C of 15g (0.037mol), and after being cooled to 63 DEG C, phosphorus trichloride 0.025mol (2.2mL) is slowly added dropwise, After being added dropwise, continue 110 DEG C of reaction 2h, 30mL concentrated hydrochloric acid is then added, flow back 6h, and reaction terminates, and is cooled to room temperature, by gained Reaction solution pours into 6 DEG C of ethyl alcohol, there is white solid precipitation, filters, and vacuum drying obtains the compound Ⅸ.
1H-NMR(500MHz,D2O)7.38(s,1H,IMZ-H),7.13(s,1H,IMZ-H),4.50(s,2H,N–CH2), 2.51(m,2H,CH2),1.35(s,9H,CH3) .ESI-MS, m/z (%): 327=[M-H+],Anal.Calc.for C9H18N2O7P2(%): C, 32.94;H,5.53;N,8.54.Found:C,33.12;H,3.69;N,8.31.
Experimental example
One, the induction and identification of osteoclast (osteoclast, OC)
Osteoclast is obtained according to following induction principles and identification method
Induce principle: pluripotential hemopoietic stem cell of the osteoclast in marrow.Pluripotential hemopoietic stem cell is first in M- Macrophage system clonogenic unit, as osteoclast precursor are divided under the action of the factors such as CSF, later further in RANKL Etc. under the stimulation of the factors fusion form multicore and the osteoclast with bone resorption function.RAW264.7 cell is small mouse Osteoclast precursor collects mouse ascites after Abelson mouse leukemia virus induction BALB/c mouse generation tumour The cell strain that mononuclear macrophage obtains is considered representing the osteoclast precursor cells in early differentiation stage.
Mainly there are three indexs for the identification of osteoclast: (1) cell nuclei is greater than or equal to 3;(2) cell TRAP enzyme contaminates Color is positive;(3) absorption lacuna can be formed in cell attachment and bone matrix surface or hydroxyapatite substrate, this is that judgement is broken Osteocyte has " goldstandard " of bone resorption function.It is generally acknowledged that cell, which has the condition of front two, just regards as osteoclast sample Cell (Osteoclast-like cells).
1, nuclear factor-kB receptor activation factor ligand RANKL inducing mouse mononuclear macrophage leukaemia cell RAW264.7 is divided into osteoclast
Material: RAW264.7 cell is purchased from Chinese Academy of Sciences Shanghai cell institute.
Reagent: nuclear factor-kB receptor activation factor ligand RANKL is purchased from Santa Cruz;α-MEM and FBS are purchased from Gibco;BSA is purchased from green skies Bioisystech Co., Ltd.
The specific steps of induction:
Induce the preparation of liquid: 10 μ g RANKL are dissolved in the PBS that 1ml is 0.1%BSA containing mass concentration, and α-MEM culture medium is dilute It releases to final concentration of 50ng/ml RANKL.
Fiber differentiation: by 264.7 cell of RAW according to 104A/hole is laid on 24 orifice plates, and induction liquid culture is added afterwards for 24 hours.Often Two days one subcultures of replacement.First day is the osteoclast formation stage, and the 4th day is the stage of ripeness.
As a result as shown in Figure 1a, RAW264.7 cell is in adherent growth when not inducing, and form is more uniform, mostly round, Polygonal, minority are spindle shape, and cell volume is smaller, have no that apocyte exists;As shown in Figure 1 b, when inducing for 24 hours, in the visual field Round and polygonal cell proportion increases, spindle shape Leukopenia, has no that apocyte generates;As illustrated in figure 1 c, 48h is induced When, cellular morphology starts to become irregular, and round mononuclearcell is in the great majority, individual protruding pseudopodium of endochylema;Such as Fig. 1 d It is shown, when inducing 72h, endochylema protruding pseudopodium in part in the visual field, it is seen that a small amount of multiple nucleus generate (cell nucleus number 3-5 It is a), it is big polymorphic irregular;As shown in fig. le, until after induction 96h, the visible apocyte to come in every shape is generated in the visual field (cell nucleus number 3-20), nucleus is gathered in cytoplasm in cluster or more clusters, and cell edges are rough, is in fried egg or burr Shape, endochylema contain more vacuole.
2, the identification of OC
Material: the osteoclast obtained in above-mentioned steps;
The specific steps of identification:
(1) TRAP is dyed
Kit: TRACP&ALP double-stain kit TaKaRa, MK300
Colouring method: being added 10 ml deionized waters inside every bottle acid phosphatase (ACP, No. 3), 1 milli is then added Sodium tartrate is risen, concussion dissolution obtains EWNN solution;Culture medium is discarded, liquid is fixed, fixes 5 minutes;Sterile water is added Fixer is diluted, discards, adds sterile water, discard;The EWNN solution prepared is added, 37 DEG C are incubated for 15~45 minutes; Dye liquor is discarded, is washed with deionized water three times;Microscopically observation is taken pictures.
As a result such as Fig. 2 a shows that wherein Fig. 2 a- (1) is to induce without RANKL, and Fig. 2 a- (2) is after RANKL is induced Foring the huge TRAP of a large amount of multicores (osteoclast markers characteristic), (black arrow refers to stained positive osteoclast-like cell Show place), volume increases, zymophore pinkiness or red (blackspot is shown as in this artwork master), and graininess is uniformly divided It is distributed in cell cytosol, and the height of the enzymatic activity of the depth reflection TRAP of color, pseudopodium is clear, and nucleus is negative, and mostly 3 Or 3 with coker.
(2) actin ring dyes
Phalloidine is stored liquid and prepared: 1mg FITC- phalloidine (Sigma) is dissolved in 1ml anhydrous methanol, is made into 1mg/ml Store liquid.Packing freezes in -20 DEG C, and drying is kept in dark place.
Colouring method: cell climbing sheet is grown 24-48 hours;Pre-temperature PBS is cleaned cell 2 times, every time 10 minutes;4% poly Formaldehyde room temperature fixes 10 minutes, and PBS is cleaned cell 3 times;0.1%Triton X-100/PBS room temperature rupture of membranes 3-5 minutes, PBS was clear It washes cell 3 times;2.5 μ l FITC- phalloidines storage liquid, which is added in 500ul PBS, is made into working solution (5 μ g/ml) and to contaminate Cell, room temperature dyes 60 minutes in closed wet box;PBS is cleaned cell 3 times;1ug/ml DAPI dyes 5min;It is extra to suck Moisture, adds fluorescence mounting liquid (neutral or meta-alkalescence buffer adds equivalent glycerol) mounting, and fluorescence microscopy is taken pictures under the microscope.
As a result such as Fig. 2 b shows that wherein Fig. 2 b- (1) is to induce without RANKL, and Fig. 2 b- (2) is after RANKL is induced A large amount of multicore megabacteriums are formd, after FITC- phalloidin, it is seen that fibroid actin ring is (mature osteoclastic thin The mark of bone resorption activity after born of the same parents are activated), structure in a ring, distribution is clear and regular, shows the normal appearance of cell, carefully Karyon is by DAPI indigo plant dye, Bian Ju, cell membrane obscure boundary, it is seen that have pseudopodium and fold.It is possible thereby to judge the shape of osteoclast At.
Two, 2- alkyl substituted imidazole base double phosphinic acid compounds Pharmacodynamics screenings and evaluation
1, inhibited proliferation of the mtt assay detection BPs to RAW264.7
(1) preparation of various concentration drug solution
The 2- methyl substituted imidazole base double phosphinic acid compounds for taking zoledronic acid (ZL) 1mmol (27.2mg), embodiment 1 to prepare (MIDP) 2- ethyl substituted imidazole base double phosphinic acid compounds (EIDP) 1mmol prepared by 1mmol (28.6mg), embodiment 2 2- propyl substituted imidazole base double phosphinic acid compounds (PIDP) 1mmol (31.4mg) of (30mg), embodiment 3 preparation, embodiment 4 are made Standby 2- normal-butyl substituted imidazole base double phosphinic acid compounds (BIDP) 1mmol (32.8mg), is dissolved in respectively in 100mL water, concussion It shakes up.Solution 1mL is taken, culture medium is added to be diluted to 5mL, as 200 μM of solution.200 μM of solution that 2.5mL is above-mentioned are taken, with culture Base is diluted to 5mL, as 100 μM of solution, and so on, proportional diluted, obtain 0.78125,1.5625,3.125,6.25, 12.5,25,50,100,200 μM of drug solution.
(2) mtt assay measures toxicity of compound
Inoculation is in the cell of logarithmic growth phase in 96 orifice plates, and cell implantation concentrations are 1.5 × 104mL-1.Blank is set 100 μ L cell suspensions are added in control group, remaining every hole.At 37 DEG C, contain 5%CO2Incubator in after overnight incubation, fractions tested The position the 2- alkyl substituted imidazole base double phosphinic acid compounds solution of the embodiment 1-4 preparation of 100 μ L various concentrations, control group are not added 100 μ L culture solutions are added, every group sets 8 parallel holes.After cultivating 72h, 20 μ L MTT (5mg/mL PBS) solution are added, are put into training It supports after continuing to cultivate 4h in case, supernatant is carefully sucked out, 150 μ L dimethyl sulfoxides, concussion 10min to bottom hole purple crystal is added It is completely dissolved, measures each hole absorbance OD490 value with enzyme linked immunological microplate reader.The hundred of control group OD value are accounted for experimental group OD value Divide than indicating cell viability.
As a result as shown in figure 3, the position the 2- alkyl substituted imidazole base two banks of zoledronic acid (ZL), embodiment 1-4 preparation Closing object can inhibit the proliferation of RAW264.7, and be in concentration dependent, but be generally less than classical bisphosphonates medicine The effect of object zoledronic acid Inhibit proliferaton.
2, non-toxic concn of the 2- alkyl substituted imidazole base double phosphinic acid compounds to RAW264.7
In order to carry out following experiment, especially for the experiment of osteoclast formation, embodiment 1-4 system is filtered out The standby position 2- alkyl substituted imidazole base double phosphinic acid compounds are necessary the non-toxic concn of RAW264.7, can exclude so real The influence that the position a 2- alkyl substituted imidazole base double phosphinic acid compounds for 1-4 preparation inhibit its precursor survival ability is applied, more preferably Ground is clearly to the effect during osteoclast formation.
According to it is in above-mentioned " mtt assay detects BPs to the inhibited proliferation of RAW264.7 " as a result, the above zoledronic acid with And the concentration ranges of 2- alkyl substituted imidazole base double phosphinic acid compounds: (1) ZL 0-3.125 μM, as shown in fig. 4 a;(2)MIDP 0-6.25 μM, as shown in Figure 4 b;(3) EIDP 0-6.25 μM, as illustrated in fig. 4 c;(4) PIDP0-12.5 μM, as shown in figure 4d; (5) BIDP 0-50 μM does not cause significantly to influence on the existence of RAW264.7 as shown in fig 4e, so after being finely adjusted i.e. It can carry out following osteoclast formation experiment.
3, the influence that the 2- alkyl substituted imidazole base double phosphinic acid compounds form OC
(1) counting method:
The cell nuclei of osteoclast is greater than or equal to 3, and osteoclast is in the anti-tartaic acid phosphoric acid of specificity The enzyme dyeing positive (TRAP+).To treated, RAW264.7 cell carries out TARP dyeing to this method, then counts under the microscope Cell nuclei is greater than or equal to 3 cell.
As a result as shown in figure 5, carrying out the osteoclast formation that BPs induces RANKL with the non-toxic concn section filtered out The experiment of influence finds that influence of the 2- different alkyl substituted imidazole base double phosphinic acid compounds to osteoclast formation is It is different, wherein PIDP (as shown in Figure 5 a), BIDP (as shown in Figure 5 b) can the inhibition OC of concentration dependent formed, especially PIDP (as shown in Figure 5 a) can significantly inhibit OC under lower concentration (1.5625,3.125 μM) and be formed;And ZL (such as Fig. 5 c It is shown), MIDP (as fig 5d), EIDP (as depicted in fig. 5e) although have relatively stronger inhibition RAW264.7 cells survival Ability, but at low concentrations to OC formation do not make significant difference.
(2) enzyme linked immunosorbent assay:
For TRAP in high expression, this experiment is to utilize Tartrate resistant acid phosphatase (TRAP) detection kit in osteoclast (green skies Bioisystech Co., Ltd) is active come the TRAP for detecting osteoclast.
Standard working solution and colour developing working solution are prepared according to the requirement of kit, takes appropriate cell or tissue lysate, it is low Fast centrifuging and taking supernatant, -80 DEG C freeze.In 96 orifice plates be arranged blank control, standard items, sample to be tested, solution in sequence according to Secondary addition, and pay attention to avoiding generating bubble.The dosage of standard items is respectively 4,8,16,24,32,40 μ L, and sample to be tested directly adds 40μL.It mixes gently, is incubated for 30min, every hole is added terminate liquid and terminates reaction.Detect light absorption value at 405nm.
As a result as shown in Figure 6, it is seen that PIDP can inhibit the TRAP activity of RAW264.7 cell, and be in concentration dependent; This experiment, which further demonstrates PIDP, has the performance for inhibiting osteoclast formation.
4, influence of the 2- alkyl substituted imidazole base double phosphinic acid compounds to OC actin ring structure
Step is with above-mentioned the induction and identification of osteoclast (osteoclast, OC) " one, ", the difference is that cell is The processed cell of PIDP.By 264.7 cell of RAW according to 104A/hole is laid on 24 orifice plates, at 37 DEG C, contains 5%CO2Incubator After middle overnight incubation, it is separately added into (0 μM, 1.5625 μM, 3.125 μM, 6.25 μM of the PIDP solution of 500 μ L various concentrations With 12.5 μM), induction liquid culture is added in culture afterwards for 24 hours.Every two days one subcultures of replacement.FITC- Phallus is used after induction 4 days Cyclic peptide carries out actin ring Coloration experiment.
As a result as shown in fig. 7, with control group completely clearly compared with actin ring, after various concentration PIDP processing Osteoclast actin ring by different degrees of destruction, cell appearance shrinkage disappears along with osteoclast pseudopodium, And these phenomenons with the raising of concentration it is all the more obvious, even can't see completely in ring at maximum concentration (12.5 μM of PIDP) The actin ring of shape structure.These results explanation, PIDP can destroy the structure of actin ring in concentration dependent, and have It may inhibit the activity of the osteoclastic bone resorption thus mediated, or even cause osteoclast cell death.
Three, the mechanism of drug action of PIDP
1. influence of the non-toxic concn PIDP to the formation of the RANKL OCs induced
Step is with above-mentioned the induction and identification of osteoclast (osteoclast, OC) " one, ", the difference is that cell is The processed cell of PIDP.By 264.7 cell of RAW according to 104A/hole is laid on 24 orifice plates, at 37 DEG C, contains 5%CO2Incubator After middle overnight incubation, it is separately added into the PIDP solution (0 μM, 3.125 μM, 6.25 μM and 12.5 μM) of 500 μ L various concentrations, Induction liquid culture is added in culture afterwards for 24 hours.Every two days one subcultures of replacement.Using TRAP staining kit to thin after induction 4 days Born of the same parents carry out TRAP dyeing.As a result as shown in Fig. 8 a-8e, under the action of non-toxic concn PIDP, the huge TARP (osteoclast of multicore Markers characteristic) stained positive osteoclast-like cell significantly reduces, i.e., the granular TRAP enzymatic activity of aterrimus in endochylema Position tails off, lighter, is indicated in figure with elliptic region.Illustrate the 2- propyl substituted imidazole base two banks chemical combination of non-toxic concn Object PIDP inhibits the osteoclast formation of RANKL induction.
2.PIDP forms the influence of related gene expression to OCs
Using RT-PCR method (reference literature Int J Mol Med.2016Mar;37 (3): 649-58.) it detects through difference The step of expression of related gene in the processed cell of the PIDP of concentration, the PIDP of the various concentration handles cell, is same as above.
As a result as shown in figure 9, MMP-9, Cathepsin K, c-Fos, NFATc1, RANK etc. are osteoclast formation phases Close marker.The combination of RANKL and RANK can activate NFATc1 by c-Fos.NFATc1 is the important tune of osteoclast formation The factor is saved, the expression of a variety of osteoclast formation marker genes such as TRAP, cathepsin K, MMP-9 is adjusted.This experiment Show that RANKL can cause the osteoclasts shape such as MMP-9, Cathepsin K, c-Fos, NFATc1, RANK in RAW264.7 cell It expresses and increases at the mRNA level in-site of Research of predicting markers gene, PIDP can then reduce the expression of these genes, illustrate that PIDP can pass through C-Fos caused by RANKL and NFATc1 expression is reduced to increase to inhibit osteoclast formation.
Influence of the 3.PIDP to OCs bone resorption related gene expression
Using RT-PCR method (reference literature European Journal of Pharmacology, 786 (2016) 60- 71.) expression of related gene in the processed cell of PIDP through various concentration is detected, the PIDP of the various concentration is processed Cell the step of be same as above.
The results are shown in Figure 10, and TRPV5 is contained in OC, may participate in the transport for the calcium being degraded in Bone resoiption pit, for bone It absorbs extremely important.Integrin β 3 is OC specific gene, is played an important role during regulating and controlling osteoclastic bone resorption.This reality It tests and shows that RANKL can cause the mRNA level in-site expression of TRPV5 in RAW264.7 cell, integrin β 3 to increase, PIDP can then drop The expression of these low genes illustrates that PIDP may press down by reducing the expression of TRPV5 and integrin β 3 caused by RANKL raising The bone resorption function of osteoclast processed.
4.PIDP forms the influence of correlative protein expression to OCs
Using Westernblot method (reference literature Bone.2010Mar;46 (3): 724-31.) it detects through various concentration The processed cell of PIDP in GAP-associated protein GAP expression, the step of PIDP of the various concentration processed cell is same as above.
As a result as shown in figure 11, c-Fos, NFATc1 are osteoclast formation Research of predicting markers.This experiment shows RANKL can cause the expression of the osteoclast formations GAP-associated protein GAP such as c-Fos, NFATc1 in RAW264.7 cell to increase, and PIDP is then The expression that these albumen can be reduced illustrates that PIDP can inhibit by reducing c-Fos caused by RANKL and NFATc1 expression raising Osteoclast formation.
Influence of the 5.PIDP to osteoclast formation associated signal paths
By 264.7 cell of RAW according to 4 × 104A/hole is laid on 6 orifice plates, at 37 DEG C, contains 5%CO2Incubator in cultivate After overnight, the induced medium of the RANKL containing 50ng/ml is added, experimental group is added 12.5 μM of the PIDP solution, trains respectively Support 0,5,15, after 30min, with RIPA (in) lysate (green skies Bioisystech Co., Ltd) lytic cell, use Westernblot method (reference literature Bone.2010Mar;46 (3): 724-31.) detect associated signal paths albumen in cell Expression.
As a result as shown in figure 12, RANKL can activate a variety of signaling molecules relevant to osteoclast formation, as Akt, p38, JNK, ERK, NF- κ B etc..This experiment shows that RANKL can cause p-p38, p-JNK, p-ERK, p-Akt in RAW264.7 cell Increase and I κ B- α decline, PIDP then can inhibit the increase of p-JNK, p-Akt, illustrates that PIDP can be by inhibiting p- caused by RANKL JNK, p-Akt expression increase to inhibit osteoclast formation.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or It changes still within the protection scope of the invention.

Claims (4)

1. a kind of 2- alkyl substituted imidazole base double phosphinic acid compounds exist preparing the application in osteoclast inhibitors, feature In the compound has the structure as shown in formula (I):
Wherein, R1For propyl or normal-butyl.
2. application according to claim 1, which is characterized in that the R1For n-propyl.
3. a kind of osteoclast inhibitors are with 2- alkyl substituted imidazole base double phosphinic acid compounds of any of claims 1 or 2 Effective component.
4. osteoclast inhibitors according to claim 3, which is characterized in that with 2- alkane of any of claims 1 or 2 Base substituted imidazole base double phosphinic acid compounds selectively add clinic made of customary adjuvant according to common process for effective component Acceptable preparation.
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