CN107629016A - Azo-Blue complex and its preparation method and application - Google Patents

Azo-Blue complex and its preparation method and application Download PDF

Info

Publication number
CN107629016A
CN107629016A CN201711110358.XA CN201711110358A CN107629016A CN 107629016 A CN107629016 A CN 107629016A CN 201711110358 A CN201711110358 A CN 201711110358A CN 107629016 A CN107629016 A CN 107629016A
Authority
CN
China
Prior art keywords
solution
azo
compound
blue
obtains
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201711110358.XA
Other languages
Chinese (zh)
Other versions
CN107629016B (en
Inventor
陈小元
郎立新
牛刚
田蕊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shamu (shanghai) Biological Technology Co Ltd
Original Assignee
Shamu (shanghai) Biological Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shamu (shanghai) Biological Technology Co Ltd filed Critical Shamu (shanghai) Biological Technology Co Ltd
Priority to CN201711110358.XA priority Critical patent/CN107629016B/en
Publication of CN107629016A publication Critical patent/CN107629016A/en
Application granted granted Critical
Publication of CN107629016B publication Critical patent/CN107629016B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B35/00—Disazo and polyazo dyes of the type A<-D->B prepared by diazotising and coupling
    • C09B35/02—Disazo dyes
    • C09B35/021—Disazo dyes characterised by two coupling components of the same type
    • C09B35/027—Disazo dyes characterised by two coupling components of the same type in which the coupling component is a hydroxy-amino compound
    • C09B35/029—Amino naphthol

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The present invention provides a kind of Azo-Blue derivative, and as shown in following formula (I), its structure contains two seralbumin conjugated groups and a Macrocyclic polyamine class chelation group NOTA.The present invention also provides the mark complex that the Azo-Blue derivative obtains after radioisotope labeling.Mark cooperation substance markers of the present invention are simple, and biology performance is good；Biological results show that it has the performance for distinguishing sentinel lymph node and secondary lymphoid knot.Purposes the invention further relates to the preparation method of the complex and its in the organ of human or animal and tissue developer is prepared.

Description

Azo-Blue complex and its preparation method and application
Technical field
The present invention relates to a kind of radioactivity complex, its preparation method and the complex as mammary sentinel lymphoscintigraphy Agent and the application of blood pool imaging agent.
Background technology
The transfer and diffusion of solid tumor generally pass through lymphatic vessel, tumor cell secretion Vascular ar endothelial growth facor-c (such as VEGF-C And VEGF-D) stimulate borderline tumor and be internally formed new lymphatic vessel, and then promote tumour cell by lymphatic vessel to lymph node Diffusion.One-level lymph node is that sentinel lymph node (Sentinel Lymph Nodes, SLNs) is the first of metastases and diffusion Stand, therefore, metastases whether have occurred to determine the grade malignancy of tumour clinically by detection sentinel lymph node (SLNs) With corresponding treatment means.At present, it is the goldstandard for judging metastases that SLNs, which organizes biopsy results, its turn into decision-making some The important evidence of the operation plan of tumour patient, including breast cancer, melanoma, colorectal cancer and prostate cancer etc..At present, The most frequently used sentinel lymph node detection method is to inject the sulfur colloid of radionuclide 99mTc marks first, with gamma detectors SLNs approximate location is primarily determined that, vital stain (vital stain of such as patent blue) is then injected after a few hours, because SLNs positions are enriched in for macroscopic vital stain, carry out extracing SLNs progress biopsies in art so as to guide.But This method has with more limitation.First, it is necessary to inject different probes at twice, adds the complexity of clinical diagnosis Property；Relative sensitivity is low when second, gamma detector position to 99mTc, and local resolution is low；3rd, blue dyes leads to Skin and adipose tissue can not often be passed through, it is therefore necessary to which peeling off SLNs peripheral adipose tissues could carry out accurately extracing SLNs；The Four, colloid is enriched in lymph node position and causes inflammatory reaction, and degradability is relatively low.Meanwhile using current detection means SLNB four clinical testing data synthesis displays are carried out, false negative rate (loss) is 4.6%-16.7%.In addition, dyestuff is noted May be by operative region colors blue when penetrating.Also there are some progress, such as 99mTc- in terms of SLNs probe exploitation at present Either coloured signal can be injected EB joints radioactivity simultaneously or near infrared fluorescent dye such as ICG is independent or joins Conjunction nano particle is imaged the detection for SLNs.Wherein diameter about 7nm extra small nano silicon particles Cornell dots (C- Dots) ratified by FDA to be used for optical detection or optics-PET bimodal detection platforms.Due to needing to meet to perform the operation simultaneously Monitoring in preceding evaluation and operative process, can typically use different imaging mode joint-detections.Therefore, these methods are still Larger limitation be present.In addition, current probe has the problem of common, it can pass through lymphatic vessel in neoplasm in situ injection One-level, secondary lymphoid knot are rapidly migrated to, and when SLND is performed the operation, the probe signals of secondary lymphoid knot can be to preceding The positioning and confirmation of whistle lymph node produce interference, so as to increase operating difficulty.
Human serum albumins (Human serum albumin, HSA) is abundance highest albumen in human plasma.Due to it Half-life in vivo is grown, and often can be used for medicine as pharmaceutical carrier with the property of the materials such as reversible combination delivery small molecule Delivery platform, for diabetes, tumour, rheumatic arthritis and infectious diseases etc..HSA also by directly as image probe, It is used for optical imagery after marked fluorescent dye, is used for PET imagings after marked radionuclide, marks Gd3+For magnetic resonance Imaging.In the research of early stage, applicant is creative to her using Azo-Blue (EB) and sero-abluminous high-affinity Wen's indigo plant obtains Azo-Blue derivative (NEB) after being transformed, it has excellent biological property as blood pool imaging agent, and The characteristics of preparation method is simple, cost is low, have broad application prospects (Chinese invention patent 201310157168.9).Separately Outside, in addition to for blood pool imaging agent, by mixed EB with18F-AlF-NEB carries out local injection, can be accurate using PET detections Really clearly SLNs is positioned, positioning reference is provided for operation.EB after albumin combination in vivo with launching fluorescence simultaneously (transmitted wave is about in 670nm), so as to carry out detection positioning with fluorescence imaging simultaneously.In addition, EB dyestuffs sheet is as blueness, Target lymph node can be dyed to blueness in surgical procedure, to confirm the accuracy of operation.This PET, fluorescence, visible three mould of naked eyes The SLNs imaging modes of state, are verified in animal model with outstanding imaging effect, and due to its security compared with High, easy katabolism, without double injection the features such as so that it has more advantage than traditional SLNs detection means.For SLNs Detection provide a kind of new simple, safe and effective method (In vivo albumin labeling and lymphatic imaging.Proc Natl Acad Sci U S A.2015；112(1):208-13).But because NEB expands Speed is dissipated, diffuses to sentinel lymph node and secondary lymphoid knot in the short time after local injection, so as to the two level in operation Lymph node can produce interference to the signal of sentinel lymph node, and increase surgical doctor differentiates the difficulty of sentinel lymph node.Meanwhile this is asked Topic and the at present common issue present in other SLNs developing methods.
The content of the invention
Based on above-mentioned background, the present invention proposes a kind of new Azo-Blue derivative, and its feature is can HSA with two Conjugated group, there is the affinity stronger with HSA, HSA-N (EB) can be formed in combination with two HSA molecules2- HSA dimerization Body, so as to which it has longer half-life period in peripheral blood, migration velocity is slower in lymphatic vessel, can distinguish sentinel lymph node and Secondary lymphoid knot, and have higher intake enrichment in tumour, have for blood pool imaging agent and sentinel lymph node developer More preferable biological property, it is expected to clinically be able to popularization and application.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
First, there is provided one kind has the radiolabeled Azo-Blue complex of excellent imaging performance；
Another object of the present invention is to provide the method for preparing described radiolabeled Azo-Blue complex；
Another object of the present invention is to provide described complex and shown preparing sentinel lymph node, pool and tumour blood pool As the application in agent.
Realize that the above-mentioned primary and foremost purpose technical scheme of the present invention there are two aspects of following part synthesis and radioactive label.
On one side, the invention provides a kind of Azo-Blue derivative (compound N (EB)2), described compound N (EB)2Shown in structure such as following formula (I)：
Wherein, R can be linking group of the structure as shown in following formula (II),
X in formula (II) is selected from C atoms or N atoms；Described n1、n2And n3It can be the same or different, take 0-3 Integer；Described Y1、Y2And Y3It can be the same or different, be selected from any one following structure：
In the solution of the present invention, when the X in the formula (II) is the C atomic time, described Y1It is preferred thatDescribed Y2 And Y3It is preferredDescribed n1It is preferred that 2, described n2And n3Preferably 0；I.e. described R structures such as following formula (III) institute Show：
Therefore, when the X in formula (II) is the C atomic time, a preferable compound N (EB) of the invention2Structure such as following formula (IV) shown in：
In the solution of the present invention, when the X in the formula (II) is the N atomic time, described Y1And Y2It is preferred Described Y3It is preferred thatDescribed n1、n2And n3Preferably 1；Shown in i.e. described R structures such as following formula (V)：
Therefore, when the X in formula (II) is the N atomic time, presently preferred compound N (EB)2Structure such as following formula (VI) shown in：
On this basis, the present invention further provides prepare compound N (EB) shown in formula (IV)2Method, it is including following Step：
1. tertbutyloxycarbonyl (Boc), the amino diazonium of the other end are introduced on the amino of 3,3'- dimethylbenzidines one end Change reaction, generate diazol；Then coupling reaction occurs with 1- amino-8-naphthol -2,4- disulfonates sodium；Remove Boc protections After base, amino and ammoniacal liquor, midbody compound A of the carbon disulfide reaction generation with thiocyanogen of deprotection；
2. 6- fluorenylmethyloxycarbonyls amino (Fmoc) -2- tertbutyloxycarbonyls (Boc) aminocaproic acid and step 1. in coupling it is anti- Fmoc protection groups, the amino and NOTA-OBu of deprotection are sloughed after answering product acylated condensationtThrough acylated condensation reaction, then take off again The Boc protection groups gone in condensation product, obtain the midbody compound B with NOTA groups；
3. 2. midbody compound B that 1. midbody compound A that step obtains and step obtain reacts, make thiocyanogen with Amino condensation reaction forms thiocarbamide key, obtains a kind of N (EB) described in formula (IV) of the present invention2。
The method of described preparation formula (IV) compound, preferably specifically includes following steps：
1) di-tert-butyl dicarbonate reaction is added dropwise in compound 1 (3,3'- dimethylbenzidines) solution of dichloromethane And purify and obtain flaxen compound 2；Under condition of ice bath, HCl and NaNO is added dropwise successively in the acetonitrile solution of compound 22Instead The solution of diazenium compound 3 of yellow should be generated；
2) in NaHCO3In 1- amino-8-naphthol -2,4- disulfonate sodium ice water solutions, the change that step 1) obtains is added dropwise The solution of compound 3, reaction obtain compound 4；Compound 4 further obtains compound 5 with trifluoroacetic acid hybrid reaction；By compound 5 are dissolved in DMF and ammoniacal liquor are added dropwise after reaction overnight；Add CS2Reaction obtains the crude product of compound 6；In acetonitrile solution Middle compound 6 and Pb (NO3)2React and purify to obtain compound 7；
The synthetic route of above step is as follows：
3) in DMF solution, compound 5 and 6- fluorenylmethyloxycarbonyls amino (Fmoc) -2- tertbutyloxycarbonyls (Boc) amino oneself Acid (compound 8), HATU and DIPEA hybrid reactions and purifying obtain compound 9；Further in DMF solution, in compound 9 Piperidines is added dropwise to react and purify to obtain compound 10；Continue in DMF solution, compound 10, NOTA-OBut, HATU and DIPEA Hybrid reaction and purify obtain compound 11；Compound 11 and trifluoroacetic acid react and purify to obtain compound 12；Specific synthesis Route is as follows：
4) compound 7, the step 3) that step 2) obtains obtain compound 12 and HATU and DIPEA hybrid reactions simultaneously purify Obtain target compound 13；Specific synthetic route is as follows：
Other N (EB) in the present invention program2The preparation method of compound is similar with the preparation method of compound 13, substantially On may be referred to the synthetic route of compound 13 and prepared.
Second aspect, the present invention further provides a kind of radiolabeled Azo-Blue complex, it is with this hair Bright described formula (I) compound N (EB)2The complex obtained for part, mark radionuclide.
In radiolabeled Azo-Blue complex of the present invention, described radionuclide can be selected from18F 、64Cu、68Ga、62Cu、67Cu、86Y、89Zr、90Y or177Lu etc., preferably18F or64Cu。
The present invention further provides the method for preparing described radiolabeled Azo-Blue complex：I.e. described puts Penetrating property nucleic and formula (I) compound N (EB)2React under proper condition, obtain described radiolabeled Azo-Blue and match somebody with somebody Compound.
Preferable preparation method is following wet method or desivac：
In a kind of preferred embodiment of the present invention, with the compound 13 shown in formula (IV) for part, wet method mark Radioactivity18F, comprise the following steps：
A1) appropriate compound 13 is dissolved in cushioning liquid or deionized water, obtains solution a；
A2) aluminium chloride is dissolved in cushioning liquid or deionized water, obtains solution b；
A3) by solution a and solution b it is well mixed after, add acetonitrile or ethanol and fresh obtained18The F ion aqueous solution, it is close Close 70~120 DEG C of 5~30min of reaction, cooling；
A4) be diluted with water step A3) after obtained reaction solution through Sep-Pak C18 chromatogram column separating purifications, with buffer solution Or water flushing chromatographic column removing is unreacted18F ion, eluted with ethanol solution hydrochloride or ethanol solution, then it is dilute through physiological saline It is sterile filtered, produces after releasing18The Azo-Blue derivative injection of F marks.
In another preferred embodiment of the present invention, with the compound 13 shown in formula (IV) for part, desivac Mark radioactivity18F, comprise the following steps：
B1) appropriate compound 13 is dissolved in cushioning liquid or deionized water, obtains solution a；
B2) aluminium chloride is dissolved in cushioning liquid or deionized water, obtains solution b；
B3) by solution a and solution b it is well mixed after, add acetonitrile or ethanol and fresh obtained18The F ion aqueous solution, it is close Close 70~120 DEG C of 5~30min of reaction, cooling；
B4) by step B3) obtained solution after aseptic filtration, is sub-packed in container, sealing of being jumped a queue after freeze-dried, When froze-dried kit；The container of packing is preferably control antibiotic bottle；
B5) to step B4) appropriate acetic acid solution or buffer solution are added in obtained medicine box, add acetonitrile or ethanol With it is fresh obtained18The F ion aqueous solution, 5~30min of closed 70~120 DEG C of reactions, cooling；
B6) be diluted with water step B5) after gained reaction solution through Sep-Pak C18 chromatogram column separating purifications, with buffer solution or It is unreacted that water rinses chromatographic column removing18F ion, eluted with ethanol solution hydrochloride or ethanol solution, then through normal saline dilution After be sterile filtered, produce18The Azo-Blue derivative injection of F marks.
In another preferred embodiment of the present invention, with the compound 13 shown in formula (IV) for part, wet method mark Remember radioactivity64Cu, comprise the following steps：
C1) appropriate compound 13 is dissolved in cushioning liquid or deionized water, obtains solution a；
C2) added in solution a64Cu2+(CuCl2) sodium acetate solution, 5~100min of closed 40~80 DEG C of reactions, cooling；
C3) step C2) partly preparation HPLC (high performance liquid chromatography) is isolated and purified resulting solution warp, and revolving removes solvent, with Phosphate buffer (PBS) or water dissolving residue, through being sterile filtered, are produced64The Azo-Blue derivative injection of Cu marks.
In another preferred embodiment of the present invention, with the compound 13 shown in formula (IV) for part, desivac Mark radioactivity64Cu, comprise the following steps：
D1) appropriate compound 13 is dissolved in cushioning liquid or deionized water, obtains solution a；
D2) by step D1) resulting solution a after aseptic filtration, is sub-packed in container, sealing of being jumped a queue after freeze-dried, Obtain froze-dried kit；The container of packing is preferably control antibiotic bottle；Situation is molded according to medicine box freeze-dried powder to may be selected in medicine box Middle increase excipient, such as mannitol, ascorbic acid etc., the dosage of adjustable nodal compound 13 and excipient, are molded medicine box Reach optimal；
D3) to step D2) gained medicine box in add appropriate acetic acid solution or buffer solution, add64Cu2+(CuCl2) Sodium acetate solution, 5~30min of closed 70~120 DEG C of reactions, cooling；
D4) step D3) partly preparation HPLC (high performance liquid chromatography) is isolated and purified resulting solution warp, and revolving removes solvent, with Phosphate buffer (PBS) or water dissolving residue, through being sterile filtered, are produced64The Azo-Blue derivative injection of Cu marks.
Used other chemical substances in above-mentioned synthesis step are commercial goods.
In the above method, the radionuclide is nuclear medicine image part, except18F and64Outside Cu, it is also possible to be67Ga 、68Ga、62Cu、67Cu、86Y、89Zr、90Y or177Lu etc.；The cushioning liquid is the material for stablizing reacting liquid pH value, can be vinegar Hydrochlorate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate and phosphate, with And their mixture etc..
Radiolabeled Azo-Blue cooperation substance markers provided by the invention are simple, and biology performance is good.Biologic test As a result show that it has the performance for distinguishing sentinel lymph node and secondary lymphoid knot, this novel performance is that current other lymph nodes show As not available for agent, sentinel lymph node developer is suitable as, is performed the operation for SLND.In addition, it is in blood In have very high intake and very long circulation time, be also suitable for blood pool imaging agent.
Brief description of the drawings
Fig. 1 AFMs characterize compound 13 and HSA combination；
Fig. 2 compounds 13 and NEB different time PET imagings figure contrasts after normal mouse tail vein injection respectively, and M- activity curve contrast during 60min after the administration of pool and bladder；
Fig. 3 compounds 13 and NEB the different time firsts and seconds lymph node PET after the injection of normal mouse foot sole pad respectively Imaging figure contrast, and m- activity curve figure during lymph node, and one-level, secondary lymphoid knot time of developing interval correlation.
Different time firsts and seconds lymph node is glimmering after the injection of normal mouse foot sole pad respectively by Fig. 4 compounds 13 and NEB Photoimaging imaging figure contrast.
Different time PET imagings image Fig. 5 compounds 13 and NEB after U87 nude mouse tumor model tail vein injections respectively Figure contrast, and the contrast of different time points tumor uptake figure.
Embodiment
Below by way of specific embodiment so that the present invention is more clearly described.
The preparation of the Azo-Blue derivative ligand (compound 13) of embodiment 1.
The synthesis of compound 2：
Put into compound 1 (3,3'- dimethylbenzidines, 4.25g, 20.0mmol) and 50mL respectively in 250mL flasks Dry dichloromethane obtains yellow solution.By 20mL di-tert-butyl dicarbonates (4.36g, 20.0mmol) dichloromethane solution It is slowly added dropwise into flask.It is stirred at room temperature down, after reacting 24h, solvent is spin-dried for obtaining yellow solid.Residue crosses silicagel column (stone Oily ether/ethyl acetate=1：3) flaxen compound 2, yield 50% are purified to obtain.1H NMR(300MHz,CDCl3)δ7.79(d, J=8.0Hz, 1H), 7.35 (d, J=8.5Hz, 1H), 7.31 (s, 1H), 7.24 (d, J=3.7Hz, 1H), 6.71 (d, J= 7.9Hz,1H),6.27(s,1H),3.63(s,2H),2.28(s,3H),2.21(s,3H),1.53(s,9H).
The synthesis of compound 3：
Under condition of ice bath, the 2.0M HCl of 15mL ice are added dropwise to 40mL compounds 2 (3.12g, 10.0mmol) second In nitrile solution.15min is stirred, then by 20mL NaNO2The ice water solution of (2.07g, 30.0mmol) is slowly added dropwise into flask, Drop continues to stir the solution of diazenium compound 3 of 30min generation yellow after finishing, stand-by.
The synthesis of compound 4:
By NaHCO3(3.36g, 40.0mmol) and 1- amino-8-naphthol -2,4- disulfonates sodium hydrate (3.19g, 10.0mmol) it is dissolved in respectively in 20mL frozen water, then the solution of diazenium compound 3 of brand-new is slowly added dropwise, continues to stir under ice bath 60min obtains purple solution.Purple solution is freeze-dried to obtain violet solid compound 4, yield 60%.1H NMR(300MHz, MeOD) δ 8.71 (s, 1H), 8.00 (dd, J=9.2,6.9Hz, 2H), 7.60 (d, J=8.6Hz, 1H), 7.53 (d, J= 18.0Hz, 2H), 7.47 (s, 2H), 7.18 (d, J=9.9Hz, 1H), 2.56 (s, 3H), 2.32 (s, 3H), 1.53 (s, 9H) .MS (LC-MS):calcd.For For C29H30N4O9S2642.1；found 641.1[M-H]-.
The synthesis of compound 5:
Compound 4 (3.22g, 5.0mmol) is thrown into the mixed solution of 20mL trifluoroacetic acids in batches, stirred at room temperature 60min.Trifluoroacetic acid is removed under reduced pressure and obtains the crude product of compound 5, yield 50%.1H NMR(300MHz,MeOD)δ8.67(s, 1H), 7.96 (d, J=9.9Hz, 1H), 7.85 (d, J=8.5Hz, 1H), 7.49 (d, J=8.7Hz, 1H), 7.43 (s, 1H), 7.28 (s, 1H), 7.24 (d, J=8.3Hz, 1H), 7.15 (d, J=9.9Hz, 1H), 6.70 (d, J=8.2Hz, 1H), 2.47 (s,3H),2.18(s,3H).MS(LC-MS):calcd.For C24H22N4O7S2542.1；found 541.1[M-H]-.
The synthesis of compound 6:
Compound 5 (1.08g, 2.0mmol) is dissolved in DMF, then 1ml ammoniacal liquor is added dropwise, lower reaction is stirred at room temperature overnight. By CS2(0.74g, 10.0mmol) is added in reaction mixture, is stirred 8 hours at 40 DEG C.Revolving removes solvent, obtains compound 6 crude product.Purified by reversed-phase column, freeze-drying obtains pure compound 6, yield 80%.
The synthesis of compound 7:
In 50mL compounds 6 (0.58g, 1.0mmol) acetonitrile solution, Pb (NO are added3)2(0.66g, 2.0mmol), It is stirred overnight at room temperature, filters, filtrate is spin-dried for obtaining crude product.Purified by reversed-phase column, freeze-drying obtains pure compound 7, produces Rate 95%.
The synthesis of compound 9:
Compound 5 (0.54g, 1.0mmol), 6- fluorenylmethyloxycarbonyls amino (Fmoc) -2- tertbutyloxycarbonyls (Boc) amino oneself Sour (compound 8) (0.43g, 1.0mmol), HATU (0.38g, 1.0mmol) and DIPEA (0.26g, 2.0mmol) are put into successively To 20mL DMF.Reactant mixture stirs 24 hours, is evaporated under reduced pressure and removes solvent, obtains crude product.By the inverted post of crude product Purifying, freeze-drying obtain pure compound 9, yield 80%.
The synthesis of compound 10:
Compound 9 (0.48g, 0.5mmol) is dissolved to 10mL DMF, and piperidines (2mL) is added dropwise under ice bath.Drop finishes, heating To room temperature, continue stirring 2 hours.Reaction mixture is evaporated under reduced pressure and removes solvent, obtains crude product.By the inverted post of crude product Purifying, freeze-drying obtain pure compound 10, yield 99%.
The synthesis of compound 11:
Compound 10 (0.37g, 0.5mmol), NOTA-OBut(0.25g,0.5mmol)、HATU(0.19g,0.5mmol) Put into successively to 5mL DMF with DIPEA (0.13g, 1.0mmol).Reactant mixture is stirred overnight, and is evaporated under reduced pressure and is removed solvent, Obtain crude product.By the inverted post purifying of crude product, freeze-drying obtains pure compound 11, yield 75%.
The synthesis of compound 12:
Compound 11 (0.24g, 0.2mmol) is thrown into the mixed solution of 20mL trifluoroacetic acids, it is small to stir 2 at room temperature When.Trifluoroacetic acid is removed under reduced pressure and obtains the crude product of compound 12.By the inverted post purifying of crude product, freeze-drying obtains pure change Compound 12, yield 80%.
The synthesis of compound 13:
Compound 7 (0.06g, 0.1mmol), compound 12 (0.10g, 0.1mmol), HATU (0.38g, 0.1mmol) and DIPEA (0.04g, 0.2mmol) is put into 2mL DMF successively.Reactant mixture is stirred overnight, and is evaporated under reduced pressure and is removed solvent, obtains To crude product.By the inverted post purifying of crude product, freeze-drying obtains pure compound 13, yield 60%.
Embodiment 2~20
Embodiment 2-20 N (EB)2Shown in compound structure such as following formula (I), wherein, R can be structure such as following formula (II) Shown linking group, with reference to listed by table 1 below, their preparation method refers to reality for the selection of each several part group in formula (II) Apply example 1：
Table 1
The radioactivity of embodiment 21.18F marks the preparation of Azo-Blue complex
1. radioactivity18The preparation of F mark freeze drying medicine boxs (exemplified by preparing 100)
The compound 13 for weighing the preparation of 10mg embodiments 1 is dissolved in 10mL 0.5mol/L NaAc_HAc buffer solution (pH=4) in, then by 0.1mg aluminium chloride (AlCl3) it is dissolved in 10mL 0.5mol/L NaAc_HAc buffer solution (pH=4) In, will the two uniformly mixing.It is sub-packed in after aseptic filtration in 100 control antibiotic bottles, is subsequently placed in freeze drier Middle freeze-drying 24 hours, sealing of jumping a queue, obtains froze-dried kit.Wanted according to medicine box yield and to constituent content in every medicine box The difference asked, its mass ratio can be made 20~100 with the dosage of modulating compound 13 and aluminium chloride：In the range of 1.
2.18F marks the preparation of Azo-Blue complex
1) wet method：In 1mL plastic tubes, 3 μ L 2mM AlCl is added3NaAc_HAc buffer solution (0.5mol/L, PH=4) and 6 μ L 3mM embodiment prepare the NaAc_HAc buffer solution of compound 13 (0.5mol/L, pH=4), then Add 0.13mL acetonitrile and about 370,000,000 Bakes (MBq)18The F aqueous solution, it is placed in boiling water and reacts 10 minutes after being sufficiently mixed. 10mL volumes are diluted with water to after reaction solution cooling, are isolated and purified through Varian Bond Elut C18 chromatographic columns (100mg), with It is unreacted that 10mL water rinses chromatographic column removing18F ion, eluted with the ethanol waters of 0.3mL 80% (HCl containing 1mM), in argon Ethanol is spin-dried under gas shielded, final product is dissolved in phosphate buffer (0.5mol/L, pH=7.4) and through being sterile filtered, obtains18The Azo-Blue complex of F marks, is identified using analytic type HPLC.
2) desivac：0.5mL 0.5mol/L NaAc_HAc buffer solution (pH=4) is added in froze-dried kit, All about 37~3700MBq is added after dissolving18F acetonitriles leacheate (obtains) from anion trapping column QMA, closed 120 DEG C of reactions 5min, cooling；Through Sep-Pak C18 chromatogram column separating purifications after being diluted with water, color is rinsed with 0.5mol/L phosphate buffers It is unreacted to compose post removing18F ion, eluted with ethanol solution hydrochloride or ethanol solution, then the sterile mistake after normal saline dilution Filter and produce18The Azo-Blue derivative injection of F marks.
The property representation that the Azo-Blue complex (compound 13) of embodiment 22. is combined with HSA
Compound 13 prepared by embodiment 1 is with HSA with molar concentration 1:After 1 mixing, atomic force microscope observation is prepared Sample.Use the Bioscope Catalyst atomic forces of one reverse optical microscope (IX71, Olympus, Japan) of connection Microscope (Bruker Santa Barbara, CA) shoots sample, it was observed that compound 13 can connect 2 HSA so as to be formed HSA dimers or crosslinking are polymer (Fig. 1).
Embodiment 23.18MicroPET imaging of the Azo-Blue derivative of F marks in normal mouse body
Purity, which is prepared, by the method for embodiment 21 is more than 95%18The Azo-Blue derivative (compound 13) of F marks, In normal FVB mouse, through tail vein injection 3.7MBq18F-AlF-N(EB)2Or18F-AlF-NEB is as control, Ran Hou Under isoflurane anesthesia, 0~60min enters Mobile state MicroPET imagings after administration, as a result sees Fig. 2.As a result show, chemical combination (the N (EB) of thing 132) there is higher intake in mouse pool, and compare and significantly improve with NEB, it can be used for pool and show Picture.
Embodiment 24.18The Azo-Blue derivative of F marks is imaged for sentinel lymph node
Purity, which is prepared, by the method for embodiment 21 is more than 95%18The Azo-Blue derivative (compound 13) of F marks, In normal FVB mouse, through injecting 10 μ L 0.37MBq in foot sole pad18F-AlF-N(EB)2Or18F-AlF-NEB is used as control, Then under isoflurane anesthesia, 0~60min enters Mobile state MicroPET imagings, and 90min and 120min after administration Static image, as a result as shown in figure 3, (the N (EB) of compound 132) can obtain high quality lymph node PET imaging, it is in lymph Migration velocity is slower in pipe, and sentinel has PET signal in longer time, and secondary lymphoid knot does not have, can be effective The two is distinguished.
Simultaneously as compound 13 can have stronger transmitting fluorescence, therefore compound 13 at 670nm after being combined with HSA It can be used for lymph node and vasculolymphatic fluorescence imaging.Carried out in normal mouse after injecting compound 13 in foot sole pad different The fluorescence imaging at time point, using NEB as control.Fig. 4 shows that compound 13 can clearly image to lymph node and lymphatic vessel, together When, its migration velocity in lymphatic vessel is slower, can distinguish sentinel lymph node and secondary lymphoid knot in a long time.
Therefore, compound 13 marks the imaging performance of PET imagings and fluorescent imaging bimodal after radionuclide, Yi Jiqu Divide the ability of sentinel lymph node and secondary lymphoid knot, can be used for sentinel lymph node developer.
The Azo-Blue derivative wet method mark radioactivity of the present invention of embodiment 25.64Cu
Comprise the following steps：
C1) compound 13 prepared by appropriate embodiment 1 is dissolved in cushioning liquid or deionized water, obtains solution a；
C2) added in solution a64Cu2+(CuCl2) sodium acetate solution, 5~100min of closed 40~80 DEG C of reactions, cooling；
C3) step C2) partly preparation HPLC (high performance liquid chromatography) is isolated and purified resulting solution warp, and revolving removes solvent, with Phosphate buffer (PBS) or water dissolving residue, through being sterile filtered, are produced64The Azo-Blue derivative injection of Cu marks.
Embodiment 26.64MicroPET imaging of the Azo-Blue derivative of Cu marks in U87 nude mouses
Purity, which is prepared, by the method for embodiment 25 is more than 95%64The Azo-Blue derivative (compound 13) of Cu marks. In U87 nude mice by subcutaneous knurl models, tail vein injection 3.7MBq64Cu-N(EB)2Or64Cu-NEB is being administered as control 1h afterwards, 4h, 24h and 48h carry out MicroPET imagings.As shown in figure 5, (the N (EB) of compound 132) there is higher take the photograph in tumour Take, be significantly higher than NEB, can be used for tumor developer.

Claims (10)

  1. A kind of 1. Azo-Blue derivative, shown in its structure such as following formula (I)：
    Wherein, R can be linking group of the structure as shown in following formula (II),
    X in formula (II) is selected from C atoms or N atoms；Described n1、n2And n3It is identical or different, take 0-3 integer；Described Y1、Y2And Y3It is identical or different, it is selected from any one following structure：
  2. 2. the Azo-Blue derivative described in claim 1, it is characterised in that：X in the formula (II) is C atoms, described Y1 ForDescribed Y2And Y3It isDescribed n1For 2, described n2And n3It is 0；I.e. described R structures are as follows Shown in formula (III)：
    Shown in described Azo-Blue derivant structure such as following formula (IV)：
  3. 3. the Azo-Blue derivative described in claim 1, it is characterised in that：X in the formula (II) is N atoms, described Y1 And Y2It isDescribed Y3ForDescribed n1、n2And n3It is 1；I.e. described R structures such as following formula (V) shown in：
    Shown in described Azo-Blue derivant structure such as following formula (VI)：
  4. 4. preparing the method for compound shown in the formula (IV) described in claim 2, comprise the following steps：
    1. introducing tertbutyloxycarbonyl (Boc) on the amino of 3,3'- dimethylbenzidines one end, the amino diazotising of the other end is anti- Should, generate diazol；Then coupling reaction occurs with 1- amino-8-naphthol -2,4- disulfonates sodium；After removing Boc protection groups, The amino of deprotection and ammoniacal liquor, midbody compound A of the carbon disulfide reaction generation with thiocyanogen；
    2. 6- fluorenylmethyloxycarbonyls amino (Fmoc) -2- tertbutyloxycarbonyls (Boc) aminocaproic acid and step 1. in coupling reaction produce Thing sloughs Fmoc protection groups, the amino and NOTA-OBu of deprotection after being acylated condensationtThrough acylated condensation reaction, contracting is then sloughed again The Boc protection groups in product are closed, obtain the midbody compound B with NOTA groups；
    3. 2. midbody compound B that 1. midbody compound A that step obtains obtains with step reacts, make thiocyanogen and amino Condensation reaction forms thiocarbamide key, obtains the compound shown in claim 2 formula (IV).
  5. 5. a kind of radiolabeled Azo-Blue complex, it is using formula (I) compound described in claim 1 as part mark The complex that note radionuclide obtains；Described radionuclide is preferred18F、64Cu、68Ga、62Cu、67Cu、86Y、89Zr、90Y Or177Any one in Lu；More preferably18F or64Cu。
  6. 6. prepare the method for the radiolabeled Azo-Blue complex described in claim 5, it is characterised in that will with right It is part to seek the compound shown in 2 formulas (IV), and radioactivity is marked using wet method18F, comprise the following steps：
    A1) compound shown in appropriate (IV) is dissolved in cushioning liquid or deionized water, obtains solution a；
    A2) aluminium chloride is dissolved in cushioning liquid or deionized water, obtains solution b；
    A3) by solution a and solution b it is well mixed after, add acetonitrile or ethanol and fresh obtained18The F ion aqueous solution, closed 70 ~120 DEG C of 5~30min of reaction, cooling；
    A4) be diluted with water step A3) after obtained reaction solution through Sep-Pak C18 chromatogram column separating purifications, with buffer solution or water It is unreacted to rinse chromatographic column removing18F ion, eluted with ethanol solution hydrochloride or ethanol solution, then after normal saline dilution It is sterile filtered, produces18The Azo-Blue derivative injection of F marks.
  7. 7. prepare the method for the radiolabeled Azo-Blue complex described in claim 5, it is characterised in that will with right It is part to seek the compound shown in 2 formulas (IV), and radioactivity is marked using desivac18F, comprise the following steps：
    B1) compound shown in appropriate (IV) is dissolved in cushioning liquid or deionized water, obtains solution a；
    B2) aluminium chloride is dissolved in cushioning liquid or deionized water, obtains solution b；
    B3) by solution a and solution b it is well mixed after, add acetonitrile or ethanol and fresh obtained18The F ion aqueous solution, closed 70 ~120 DEG C of 5~30min of reaction, cooling；
    B4) by step B3) obtained solution after aseptic filtration, is sub-packed in container, sealing of being jumped a queue after freeze-dried, waits until Froze-dried kit；The container of packing is preferably control antibiotic bottle；
    B5) to step B4) appropriate acetic acid solution or buffer solution are added in obtained medicine box, add acetonitrile or ethanol and new It is fresh obtained18The F ion aqueous solution, 5~30min of closed 70~120 DEG C of reactions, cooling；
    B6) be diluted with water step B5) after gained reaction solution through Sep-Pak C18 chromatogram column separating purifications, rushed with buffer solution or water It is unreacted to wash chromatographic column removing18F ion, eluted with ethanol solution hydrochloride or ethanol solution, then the nothing after normal saline dilution Bacterium is filtered, and is produced18The Azo-Blue derivative injection of F marks.
  8. 8. prepare the method for the radiolabeled Azo-Blue complex described in claim 5, it is characterised in that will with right It is part to seek the compound shown in 2 formulas (IV), and radioactivity is marked using wet method64Cu, comprise the following steps：
    C1) compound shown in appropriate (IV) is dissolved in cushioning liquid or deionized water, obtains solution a；
    C2) added in solution a64Cu2+(CuCl2) sodium acetate solution, 5~100min of closed 40~80 DEG C of reactions, cooling；
    C3) step C2) partly preparation HPLC (high performance liquid chromatography) is isolated and purified resulting solution warp, and revolving removes solvent, with phosphoric acid Salt buffer (PBS) or water dissolving residue, through being sterile filtered, are produced64The Azo-Blue derivative injection of Cu marks.
  9. 9. prepare the method for the radiolabeled Azo-Blue complex described in claim 5, it is characterised in that will with right It is part to seek the compound shown in 2 formulas (IV), and radioactivity is marked using desivac64Cu, comprise the following steps：
    D1) compound shown in appropriate (IV) is dissolved in cushioning liquid or deionized water, obtains solution a；
    D2) by step D1) resulting solution a after aseptic filtration, is sub-packed in container, sealing of being jumped a queue after freeze-dried, obtains Froze-dried kit；The container of packing is preferably control antibiotic bottle；Situation is molded according to medicine box freeze-dried powder to may be selected to increase in medicine box Add excipient, such as mannitol, ascorbic acid etc., can adjust (IV) shown in compound and the dosage of excipient, make medicine box into Type reaches optimal；
    D3) to step D2) gained medicine box in add appropriate acetic acid solution or buffer solution, add64Cu2+(CuCl2) acetic acid Sodium solution, 5~30min of closed 70~120 DEG C of reactions, cooling；
    D4) step D3) partly preparation HPLC (high performance liquid chromatography) is isolated and purified resulting solution warp, and revolving removes solvent, with phosphoric acid Salt buffer (PBS) or water dissolving residue, through being sterile filtered, are produced64The Azo-Blue derivative injection of Cu marks.
  10. 10. application of the radiolabeled Azo-Blue complex in nucleus medical imaging agent is prepared described in claim 5；Institute The preferred sentinel lymph node developer of nucleus medical imaging agent, cardiac blood pool imaging agent or the tumour blood pool imaging agent stated.
CN201711110358.XA 2017-11-12 2017-11-12 Evans blue complex and preparation method and application thereof Active CN107629016B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711110358.XA CN107629016B (en) 2017-11-12 2017-11-12 Evans blue complex and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711110358.XA CN107629016B (en) 2017-11-12 2017-11-12 Evans blue complex and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN107629016A true CN107629016A (en) 2018-01-26
CN107629016B CN107629016B (en) 2020-05-22

Family

ID=61108534

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711110358.XA Active CN107629016B (en) 2017-11-12 2017-11-12 Evans blue complex and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN107629016B (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108690119A (en) * 2018-06-04 2018-10-23 莎穆(上海)生物科技有限公司 A kind of polypeptide prodrug of Evans blue modification and its preparation and application
WO2020160222A3 (en) * 2019-01-30 2020-09-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conjugates of bivalent evans blue dye derivatives and methods of use
US10981866B2 (en) 2017-10-03 2021-04-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chemical conjugates of Evans Blue derivatives and their use as radiotherapy and imaging agents
WO2024136755A1 (en) * 2022-12-23 2024-06-27 National University Of Singapore Albumin binding compounds and methods of use thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911256A (en) * 2012-11-02 2013-02-06 厦门大学 Radioactive label polypeptide coordination complex and preparation method and application thereof
CN103242255A (en) * 2013-04-28 2013-08-14 厦门大学 Evans blue complex as well as preparation method and application thereof
CN106946899A (en) * 2017-03-21 2017-07-14 莎穆(上海)生物科技有限公司 A kind of camptothecin prodrug and its preparation and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911256A (en) * 2012-11-02 2013-02-06 厦门大学 Radioactive label polypeptide coordination complex and preparation method and application thereof
CN103242255A (en) * 2013-04-28 2013-08-14 厦门大学 Evans blue complex as well as preparation method and application thereof
CN106946899A (en) * 2017-03-21 2017-07-14 莎穆(上海)生物科技有限公司 A kind of camptothecin prodrug and its preparation and application

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10981866B2 (en) 2017-10-03 2021-04-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chemical conjugates of Evans Blue derivatives and their use as radiotherapy and imaging agents
CN108690119A (en) * 2018-06-04 2018-10-23 莎穆(上海)生物科技有限公司 A kind of polypeptide prodrug of Evans blue modification and its preparation and application
CN108690119B (en) * 2018-06-04 2022-04-19 莎穆(上海)生物科技有限公司 Evans blue modified polypeptide prodrug and preparation and application thereof
WO2020160222A3 (en) * 2019-01-30 2020-09-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Conjugates of bivalent evans blue dye derivatives and methods of use
CN113366064A (en) * 2019-01-30 2021-09-07 美国政府健康及人类服务部 Conjugates of divalent evans blue dye derivatives and methods of use
WO2024136755A1 (en) * 2022-12-23 2024-06-27 National University Of Singapore Albumin binding compounds and methods of use thereof

Also Published As

Publication number Publication date
CN107629016B (en) 2020-05-22

Similar Documents

Publication Publication Date Title
CN107629016A (en) Azo-Blue complex and its preparation method and application
AU2022228391B2 (en) Compound targeting prostate specific membrane antigen, and preparation method therefor and use thereof
CN108026144B (en) 18F-labeled inhibitors of Prostate Specific Membrane Antigen (PSMA) and their use as imaging agents for prostate cancer
CN114796528B (en) Tumor-specific targeting polypeptides and uses thereof
CN114933633B (en) Natural peptide probe for specifically recognizing FGFR4 and application thereof
CN114369084B (en) Truncated Evans blue modified fibroblast activation protein inhibitor and preparation method and application thereof
CN110845572A (en) Tumor-targeted GRP (glass-fiber reinforced Polypropylene) analogue and application thereof
CN106967152A (en) Compound that a kind of fluorine 18 is marked and preparation method and application
CN113880917B (en) Tumor high affinity peptides and application thereof
CN111675750A (en) Tumor targeting peptide aiming at carcinoembryonic antigen related adhesion molecule CEACAM and application thereof
CN110312517A (en) Luteinizing hormone releasing hormone receptor (LHRH-R) conjugates and uses thereof
CN109803973A (en) Chelate PSMA inhibitor
CN112961215B (en) Polypeptide and tumor targeting peptide, tumor detection reagent, tumor surgery navigation contrast medium and tumor targeting drug thereof
WO2024017317A1 (en) Her2 targeting peptide molecule and use thereof
CN116751258A (en) MDM2/MDMX targeting polypeptide and application thereof
CN115286693A (en) Tumor targeting peptide aiming at carcinoembryonic antigen related cell adhesion molecule CEACAM6 and application thereof
CN113912607B (en) SNAP-tag probe and preparation method and application thereof
WO2022170732A1 (en) Truncated evans blue modified fibroblast activation protein inhibitor, preparation method therefor, and application thereof
CN105884867B (en) 18The affinity body class compound and the preparation method and application thereof of F label
CN112480212A (en) High-affinity peptide of targeted hepatocyte growth factor and application thereof
CN111548305A (en) Quinoline compound for targeting PSMA (patterned middle molecular weight ligands) and preparation method thereof
CN110272539A (en) It is a kind of for combining the ligand conjugates of PMSA, probe and its preparation method and application
CN118480031B (en) Near infrared fluorescent probe for actively targeting epidermal growth factor receptor detection and preparation method and application thereof
CN115317627B (en) Application of ABT-510 peptide in preparation of tumor imaging agent
CN117777237A (en) BCMA-targeted polypeptide and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant