Technical background
Bone is a kind of special connective tissue, and sclerotin is a main component wherein, and it is made up of osteocyte, gtelatinous fibre and bone matrix again.The content of inorganic salt is very high in the bone matrix, accounts for 65%~70% of adult bone dry weight, and its main component is a hydroxyapatite.
Two phosphonic acids are a class has obvious affinity to bone chemical compounds, studies show that, calcium ion in two phosphonic hydroxyls and two phosphates and the skeleton forms the stable complex of three teeth, thereby make two phosphonic acids be oriented to the surface of bone quickly and efficiently, its uptake ratio in bone can be up to 50%~60%.Just because of this clear and definite targeting, two phosphonic acids not only are used for the treatment of osteopathia separately, also can link to each other with drug molecule, radionuclide and become the carrier of bone target medicine.
Two phosphonic acids can suppress formation, growth and the dissolving of hydroxylapatite crystal and overall material thereof, having direct inhibition osteoclast forms and bone resorption, as anti-bone resorption inhibitor, can effectively treat osteoporosis clinically, reduce the incidence rate of osteoporotic fracture.First generation bisphosphonates has appearred in the latter stage sixties, as etidronate disodium (Etidronte), clodronate disodium (Clodronate) etc., their suppress bone Reabsorption a little less than.Second filial generation bisphosphonates, as Sodium Pamidronate (Pamidronate), it suppresses the ability of bone resorption than the etidronate height, but it is bigger to the gastrointestinal side effect.Two phosphonic acids of Shang Shi the third generation in recent years, (Alendronate, ABP), its effect that suppresses bone resorption is high about 100 times than Sodium Pamidronate as Alendronic acid.
In addition,
99mTwo phosphonic acids of Tc labelling are used to the diagnosis and the treatment of bone disorder.Because
99mThe energy of of Tc is moderate, and the half-life is lacked (half-life is 6.02h), low price in addition, therefore
99mThe phosphoric acid salt of Tc labelling and bisphosphonates are applied to the bone video picture, wherein with
99mTc-MDP uses the most extensive, still
99mThe specificity of Tc-MDP is lower, and non-target tissue removes slow, and time of developing is long, and the medicine injection is long at interval with time of developing, generally reaches 2~3h.The characteristics that non-target tissues such as the developer of ideal bone should have that close bone is strong, blood is removed fast, Liver and kidney absorb is low, specificity is good etc., up to very ideal skeletal imaging agent not also at present, so to seek novel skeletal imaging agent be very significant work.
Nitric oxide (NO) is important courier's material and effector molecule, participates in numerous physiology and pathological processes in the body.Discovered in recent years, NO is the important regulatory factor of bone metabolism, not only influence the function of osteoclast, but also influence osteoblastic differentiation and propagation, the NO of debita spissitudo is maintained in osteocyte and osteoclast activity is necessary, the NO of high concentration then mainly shows as the activity that suppresses osteoclast relatively, induces the osteoclast apoptosis.Therefore, introduce a certain amount of exogenous NO, can optionally suppress the bone resorption of osteoclast, reach the osteoporotic purpose of treatment to osseous tissue.But because the complexity of NO physiological action, development targeting NO donor prodrug is extremely important.This class targeting prodrug both can discharge NO at specific position, to obtain the physiological effect of expectation, can interference body yet other NO sensitive part.In view of NO to the bone metabolism regulating action, and two phosphonic acids treatment osteoporosis effect and bone targeting, the two phosphonic acids medicines (NO-BPs) of some NO donors have been synthesized in the Lazzarato design.Because two phosphonic bone targetings, NO-BPs can realize the directed NO of release, not only reduce the side effect enhancing of NO to its hetero-organization and organ, but also two phosphonic osteoporosis curative effect (Loretta Lazzarato etc. have been improved, Synthesis of NO-Donor Bisphosphonates and Their in-Vitro Action on BoneResorption.J.Med.Chem.2005,48,1322-1329).Because NO-BPs can be from promoting bone formation, suppress two aspects reductions of bone resorption bone loss therefore have remarkable advantages than traditional osteosporosis resistant medicament.NO-Bps also can reduce the gastrointestinal side effect of existing diphosphonate, and an amount of NO can protect hepatocyte, prevents hepatocellular apoptosis, repairs effects such as hepatic injury.
Based on above achievement in research, consider two phosphonic close bone performances, osteoporosis curative effect and stronger side effect such as gastrointestinal reaction simultaneously, we design and have studied with two phosphonic acids is the NO donor medicine (NO-BPs) of bone targeting vector, and has prepared NO-BPs's
99mThe Tc coordination compound, and carried out the bio distribution experiment, preliminary experimental result shows:
99mThe coordination compound of the NO-BPs of Tc labelling, as
99mTc-NMBBDP has good bone picked-up and bone and is detained, and ratio height, the specificity of bone and non-target organ are good, are expected to become a kind of novel skeletal imaging agent.At present,
99mThe NMBBDP coordination compound of Tc labelling there is no any report at home and abroad.
Summary of the invention
The objective of the invention is to overcome the shortcoming of prior art, the synthetic novel coordination compound of a class of design with good close bone performance; And be provided for medicine box for preparing this coordination compound and preparation method thereof, and the method for such coordination compound of medicine box preparation.Make such coordination compound that obtains can be used for developer, especially skeletal imaging agent and the tumor developer of human or animal organ or tissue.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of close bone is provided
99mTc marker ligand compound is by radionuclide
99mTc and part are formed, described coordination compound with
99mThe TcO group electrically is negative charge as centronucleus, and wherein the general structure of part is as follows:
R in the formula
1Be aliphatic hydrocarbon, aromatic rings or the nitrogen heterocyclic ring that contains 1~2 NO-donor; X is-H ,-OH or-NH
2Deng group; Y is oxygen or sulfur or amino; The integer of n=2~9.
Described part can obtain by following preparation method:
A. be raw material and AgNO with halogenated carboxylic acid or hydroxy carboxylic acid
3Or HNO
3Prepared in reaction nitryl carboxylic acid; Wherein, described halogenated carboxylic acid or hydroxy carboxylic acid are to hydroxymethyl-benzoic acid, benzylbenzoic acid, 6-bromocaproic acid, 5-chloro pentane acid, 2,3-two chloro-3-benzenpropanoic acids or 4,5-two chloro pentane acids etc.;
B. above-mentioned nitryl carboxylic acid and SOCl
2Or PCI
3Reaction obtains corresponding carboxylic acid halides;
C. above-mentioned carboxylic acid halides and amino disodium phosphonate salt react under alkali condition, obtain target compound.
In the described coordination compound of the present invention, R in the preferred structure
1Coordination compound for one of following structure:
A further preferred complexes of the present invention, part is NMBBDP, R in its structure
1For
X is-OH; Y is amino; And n=3; Particular chemical as shown in the formula:
Part NMBBDP can prepare in accordance with the following methods:
A. being raw material and HNO to hydroxymethyl-benzoic acid
3Prepared in reaction 4-nitre oxygen ar-Toluic acid;
B. above-mentioned 4-nitre oxygen ar-Toluic acid and SOCl
2Reaction obtains corresponding carboxylic acid halides;
C. above-mentioned carboxylic acid halides and Alendronic acid sodium salt react under alkali condition, obtain described NMBBDP ligand compound.
Another further preferred complexes of the present invention, part is NHEBDP, R in its structure
1For
X is-OH; Y is amino; And n=3; Particular chemical as shown in the formula:
Part NHEBDP can prepare in accordance with the following methods:
A. be raw material and AgNO with the 6-bromocaproic acid
3Prepared in reaction 6-nitre oxy hexanoic acid;
B. above-mentioned 6-nitre oxy hexanoic acid and SOCl
2Reaction obtains 6-nitre oxygen caproyl chloride;
C. above-mentioned 6-nitre oxygen caproyl chloride and Alendronic acid sodium salt react under alkali condition, obtain described NHEBDP ligand compound.
Close bone of the present invention
99mThe preparation method of Tc marker ligand compound can may further comprise the steps:
Wet method labelling: described part, Reducing agent, antioxidant and excipient are followed successively by 1~50: 1~25 in molar ratio: 1~25: 20~100 wiring solution-formings, place the penicillin bottle, the pH value of solution is 4~6, gets 1~5mL
99mTcO
4Fresh leacheate injects in the bottle, shakes up, and room temperature leaves standstill that promptly to obtain putting in 10~15 minutes pure greater than 90% described close bone
99mTc marker ligand compound.
The present invention also provides a kind of described close bone that is used to prepare
99mThe medicine box of Tc marker ligand compound, this medicine box is made up of described part, Reducing agent, antioxidant, excipient and NaCl; Wherein, the mol ratio of described part, Reducing agent, antioxidant and excipient is followed successively by 1~50: 1~25: 1~25: 20~100; The preferred stannous chloride of described Reducing agent; Antioxidant preferred vitamin C; The preferred glucose of excipient.
The present invention also provides a kind of preparation method of described medicine box, may further comprise the steps:
With described part, Reducing agent, antioxidant and excipient are followed successively by 1~50: 1~25 in molar ratio: 1~25: 20~100 use the normal saline wiring solution-forming, the pH value of solution is 4~6 before the lyophilizing, after the filtering with microporous membrane sterilization with 0.22 μ m, be installed in the aseptic penicillin bottle filtrate branch, again the penicillin bottle is placed freeze dryer, the control temperature is-45~-40 ℃ of pre-freezes 2 hours, carry out vacuum drying then, naturally heating up and finally control temperature is 25~28 ℃, kept 4 hours, vacuum is 0.7Pa, fills nitrogen 1 minute again in above-mentioned lyophilizing system, and the penicillin bottle rubber stopper seal of white lyophilized powder will be housed then, the reuse aluminium lid further seals, after quality inspection is qualified, post label, place 5~8 ℃ of refrigerators to preserve; The preferred stannous chloride of described Reducing agent; Antioxidant preferred vitamin C; The preferred glucose of excipient.
Utilize above-mentioned medicine box to prepare described close bone
99mThe method of Tc marker ligand compound is as follows:
Get 1~5mL
99mTcO
4 -Fresh leacheate injects the above-mentioned medicine box for preparing, and shakes up, and after solid dissolved fully, room temperature left standstill that promptly to obtain putting in 10~15 minutes pure greater than 90% close bone
99mTc marker ligand compound.
In the chemicals of the present invention, except that described part, all the other are all from commercially available.
The present invention also provides described close bone
99mThe application of Tc marker ligand compound in preparation skeletal imaging agent and metastatic bone cancer and tumor developer.
The present invention designs that to have synthesized a class new
99mThe NO-donor diphosphonic acid complex and the medicine box thereof of Tc labelling, and announced and the preparation method of medicine box designed the method for preparing such coordination compound with wet method and easy medicine box method.Compared with prior art, the new close bone for preparing by described method
99mThe beneficial effect that the coordination compound tool of Tc labelling is following:
1. the picked-up of osseous tissue initial stage is high, the holdup time is long;
2. target/non-target ratio height, blood, liver, kidney etc. organize intake low, and its performance is better than clinical widely used at present both at home and abroad
99mThe Tc-MDP skeletal imaging agent;
3.
99mThe two phosphonic acids medicine boxs of the NO-donor of Tc labelling can be used for nuclear medicine diagnostic imaging, and in addition, the two phosphonic acids medicine boxs of NO-donor are through other nucleic, as
186Re etc. also can be used for the treatment of osteopathias such as rheumatism, rheumatoid, tumor behind the labelling.The design of such new formulation with succeed in developing, be expected in the diagnosis and treatment of illness such as osteopathia and metastatic bone cancer, play a significant role.
The specific embodiment
The preparation of embodiment 1 wet method
99mThe part of Tc labelling is the close bone coordination compound of NMBBDP
1. prepare the NMBBDP part
Synthetic route is as follows:
1.1 in the 250ml there-necked flask, add 3g (0.02mol) to cyano group benzyl chloride 2 and 50mLH
2O stirs heating for dissolving.Drip dense HCl (1.28mol, about 80ml) in three batches, backflow 5-6h is cooled to room temperature.Standing over night is regulated pH value with NaOH and is about 3~4, filters washing, solid H
2O washing three times, vacuum drying must be to hydroxymethyl-benzoic acid white solid 3;
Under the cooling of ice-water bath, in the 100ml there-necked flask, add 15mlAc
2O and 7.6mlHNO
3, stir 30min.Drip by 2g hydroxymethyl-benzoic acid (3) and 15mlAc in batches
2The molten slurry that O forms keeps in the process of dropping stirring and holding temperature is no more than 0 ℃.Reaction 3h adds 30mlH
2O, and stir 30min.Filter white solid, use H
2(3 * 15ml) washings, vacuum drying obtains 4-nitre oxygen ar-Toluic acid white solid 4 to O.
Its spectral data is:
IR(KBr,v,cm
-1):1778(-C=O),1639(v
as,NO
2),1610(Ar),1281(v
s,NO
2),858(O-N).
1H-NMR(DMSO,δ,ppm):5.30(s,2H,-CH
2),7.22(d,2H,Ar-H),7.64(d,2H,Ar-H)。
1.2 the 4-nitre oxygen ar-Toluic acid 4 of 1mmol (about 0.197g) step 1.1 preparation is joined in the there-necked flask of 100mL, stir down, drip 20mLSOCl
2, dropwise, heat up gradually and reflux 2h.Unreacted SOCl is removed in distilling under reduced pressure
2, get 4-nitre oxygen methyl benzoyl chloride yellow solid 5, drop in the next step without just being further purified.
1.3 Allan phosphoric acid ABP 1 is dissolved in the NaOH solution of 2N, and is transferred in the there-necked flask.The THF drips of solution of the 4-nitre oxygen methyl benzoyl chloride 5 of step 1.2 preparation is added in the there-necked flask, after dropwising, the about 4h of reflux.After reaction is finished, be cooled to room temperature, regulate pH to 3-4, leave standstill 2h, filter to such an extent that precipitate with 2N hydrochloric acid.Precipitation is used acetone, water, washing with acetone successively.The thick product of infrared oven dry is used H
2O-alcoholic solution recrystallization obtains pure product NMBBDP 6.
The spectral data of product is:
IR(KBr,v,cm
-1):3245(-OH),1768(-C=O),1628(v
as,NO
2),1613(Ar),1284(v
s,NO
2),1177(-P=O),924(O-N).
1H-NMR(D
2O,δ,ppm):1.89(m,4H,-CH
2CH
2-),3.30(t,2H,-CH
2),5.71(s,2H,-CH
2),7.72(d,2H,Ar-H),7.91(d,2H,Ar-H)。
2. wet method prepares close bone
99mTc marker ligand compound
The NMBBDP ligand solution 0.5mL that gets concentration and be step 1 preparation of 4mg/mL joins in the penicillin bottle, and adding vitamin C (4mg), glucose (4mg) and concentration more successively is the SnCl of 2g/L
22H
2The hydrochloric acid solution of O (0.5mL) shakes up pH to 4~6 of using the 0.1MNaOH regulation system afterwards.(activity is about 0.5~1.0mCi) to add 1mL
99mTcO
4 -Leacheate, abundant mixing, room temperature is placed 10~15min, obtains
99mTc-NMBBDP.
Detect through the lipid experiment, make
99mThe lgP=-2.48 of Tc-NMBBDP coordination compound shows
99mTc-NMBBDP is a hydroaropic substance.
Analyze its putting is pure with thin layer chromatography (TLC): be carrier with the polyamide film, developing solvent is respectively normal saline and acetonitrile, mensuration the results are shown in Table 1, use the TLC method to detect
99mThe putting of Tc-NMBBDP is pure greater than 90%.
The TLC analysis result (Rf value) of the various components of table 1
The preparation of embodiment 2 NMBBDP medicine boxs and the preparation of medicine box method
99mThe close bone coordination compound of Tc labelling
1. medicine box is made up of NMBBD part, Reducing agent stannous chloride, antioxidant vitamin C, excipient glucose and NaCl, and preparation method is as follows:
Calculate with each medicine box, various constituent mass proportionings are as follows: NMBBD part, vitamin C, glucose that embodiment 1 step 1 prepares are followed successively by 10mg, 10mg, 200mg, take by weighing each component, and mixing behind physiological saline solution, adds a certain amount of SnCl
22H
2The hydrochloric acid solution of O, regulating pH value is 4~6, aseptic filtration is sub-packed in the penicillin bottle of 10mL specification, after the lyophilization, in the sealing of vacuum condition lower cover, promptly gets the NMBBDP medicine box, and places 5~8 ℃ of refrigerators to preserve.
2. adopt the preparation of medicine box method
99mTc-NMBBDP
It is fresh to get 1mL
99mTcO
4 -Leacheate (activity is about 0.5~1.0mCi) and injects above-mentioned NMBBDP medicine box, shakes up, treat that solids dissolves fully after, room temperature leaves standstill 20min, promptly obtains
99mTc-NMBBDP.Use embodiment 1 described TLC method to detect and show what the medicine box method prepared
99mThe putting of Tc-NMBBDP is pure greater than 91%.
The preparation of embodiment 3 wet methods
99mThe part of Tc labelling is the close bone coordination compound of NHEBDP
1. prepare the NHEBDP part
Synthetic route is as follows:
1.1 0.01mol 6-bromocaproic acid 7 is dissolved in 10ml second eyeball, drop in the 20ml acetonitrile solution of 3.4g silver nitrate, 50 ℃ of holding temperatures dropwise, and are warming up to 70 ℃, reaction 6h, this course of reaction answers lucifuge to carry out.Cooling is filtered, and vacuum rotary steam removes and desolvates, and excess adds 8ml water, with ethyl acetate extraction (15mL * 3), and anhydrous sodium sulfate drying.Boil off ethyl acetate, excess adds the 50ml ether, filters.Filtrate eliminates solvent through distillation, obtains weak yellow liquid 6-nitre oxy hexanoic acid 8.
1.2 the 6-nitre oxy hexanoic acid 8 of 10mmol step 1.1 preparation is added in the there-necked flask, stir down, drip 20mLSOCl
2, dropwise, slowly be warming up to 60~70 ℃, till no HCl emits.Unnecessary SOCl is removed in distilling under reduced pressure
2, obtain 6-nitre oxygen caproyl chloride 9 crude products, directly drop into the next step.
1.3 ABP1 is dissolved in the NaOH solution of 2N, and is transferred in the there-necked flask.The THF drips of solution of the 6-nitre oxygen caproyl chloride 9 of step 1.2 preparation is added in the there-necked flask, after dropwising, reflux 4-5h.Be cooled to room temperature, regulate pH to 3, leave standstill 2h, filter to such an extent that precipitate with 2N hydrochloric acid.Precipitation is used acetone, water, washing with acetone successively, obtains thick product.The thick product of infrared oven dry is used H
2O-alcoholic solution recrystallization obtains pure product NHEBDP 10.
2. wet method prepares close bone
99mTc marker ligand compound
The NHEBDP ligand solution 0.5mL that gets concentration and be step 1 preparation of 4mg/mL joins in the penicillin bottle, and adding vitamin C (4mg), glucose (4mg) and concentration more successively is the SnCl of 2g/L
22H
2The hydrochloric acid solution of O (0.5mL) shakes up pH to 4~6 of using the 0.1MNaOH regulation system afterwards.(activity is about 0.5~1.0mCi) to add 1mL
99mTcO
4 -Leacheate, abundant mixing, room temperature is placed 10~15min, obtains
99mTc-NHEBDP.
The putting that detects 99mTc-NHEBDP through the TLC method is pure greater than 90%.
99mThe mensuration of Tc-NMBBDP vitro stability
To prepare according to embodiment 2
99mTc-NMBBDP places room temperature,, analyzes investigation in different time (1,2,3,4,5,6h) sampling with the TLC method of embodiment 1
99mTc-NMBBDP is vitro stability at room temperature.The result shows:
99mAfter Tc-NMBBDP at room temperature placed 6h, its mark rate and outward appearance did not have significant change.
99mDistribute in the body of Tc-NMBBDP
Prepare mark rate greater than 90% according to embodiment 2
99mTc-NMBBDP solution.Get that (18~22g) carry out bio distribution experiment, through tail vein injection 0.1mL (about 0.74~1.48MBq) with a collection of 30 normal kunming mices
99mTc-NMBBDP solution, respectively at injection back 5,15,30,60,120,180min the disconnected neck of two groups of mices is put to death then, get relevant organ and tissues such as blood, the heart, liver, lung, brain, muscle, bone and spleen, weigh after cleaning, survey radiocounting, calculate the intake (ID%/g) of every gram tissue, the results are shown in Table 2.
Table 2.
99mTc-NMBBDP is bio distribution (%ID/g:mean ± S.D.:n=5) in the normal mouse body
99mDistribute in the body of Tc-NHEBDP
Prepare coordination compound with method with embodiment 3
99mTc-NHEBDP, (18~22g) carry out the bio distribution experiment to get 9 normal kunming mices.Tail vein injection 0.1mL (about 0.74~1.48MBq)
99mTc-NHEBDP solution is put to death the disconnected neck of mice respectively at injection back 30,120,180min then, gets relevant organ and tissues such as blood, the heart, liver, lung, brain, muscle, bone and spleen, weigh after cleaning, survey radiocounting, calculate the intake (ID%/g) of every gram tissue, the results are shown in Table 3.
Table 3.
99mTc-NHEBDP is bio distribution in the normal mouse body
(%ID/g;mean±S.D.;n=3)
99mDistribute in the body of Tc-MDP
Get with above-mentioned body in distribute same batch of 30 normal kunming mices of experiment (18~22g), undertaken by similarity condition by the same operation personnel
99mThe animal organism of Tc-MDP distributes and tests, and tail vein injection 0.1mL (about 0.74~1.48MBq)
99mTc-MDP solution, respectively at injection back 5,15,30,60,120,180min the disconnected neck of mice is put to death then, get relevant organ and tissues such as blood, the heart, liver, lung, brain, muscle, bone and spleen, weigh after cleaning, survey radiocounting, calculate the intake (ID%/g) of every gram tissue, the results are shown in Table 4.
Table 4.
99mTc-MDP is bio distribution (%ID/g in the normal mouse body; Mean ± S.D.; N=5)
As can be seen from Table 2,
99mTc-NMBBDP is when injection 15min, 30min, 60min, and the intake of osseous tissue (ID%/g) is respectively 36.33,41.76,43.93, shows that it has good picked-up and delay in osseous tissue.In addition, corresponding time blood intake is respectively 0.93,0.40,0.19, and the liver intake is respectively 0.40,0.23,0.19, embodies good bone and non-target organ ratio, with
99mTc-MD compares,
99mTc-NMBBDP is high in the picked-up of osseous tissue initial stage, the holdup time is long, and except kidney, in other non-target tissue, especially the aggregate concentration in liver, the spleen has the reduction of highly significant, is expected to become a kind of novel skeletal imaging agent.
As can be seen from Table 3, with
99mTc-NMBBDP compares,
99mThe preliminary biological experimental data of Tc-NHEBDP shows,
99mTc-NHEBDP also has certain bone resorption, but non-target tissues' absorptions such as its liver, lung, spleen are higher comparatively speaking, and we also further explore to this.