CN107311941A

CN107311941A - 18EGFR positive electron tracers of F marks and preparation method and application

Info

Publication number: CN107311941A
Application number: CN201710409670.2A
Authority: CN
Inventors: 黄顺; 郑希; 赵肃清; 韩彦江; 吴湖炳; 王全师; 杜志云; 张焜
Original assignee: Guangdong University of Technology; Southern Hospital Southern Medical University
Current assignee: Guangdong University of Technology; Southern Hospital Southern Medical University
Priority date: 2017-06-02
Filing date: 2017-06-02
Publication date: 2017-11-03

Abstract

The invention discloses one kind¹⁸F mark EGFR positive electron tracers and its preparation method and application, structure such as formula A.Positive electron tracer of the present invention can be used for the PET tumor imaging of tumour especially EGF (EGFR) height expression, it is further used for the screening of micromolecular inhibitor class medicaments insensitive individual, the clinical value with diagnosis and monitoring treatment curative effect.Positive electron tracer preparation method of the present invention is convenient, simple, quick, and full-automatic production can be achieved, scientific research and clinical demand can be met.

Description

18EGFR positive electron tracers of F marks and preparation method and application

Technical field

The present invention relates to one kind¹⁸EGFR positive electron tracers of F marks and its preparation method and application.

Background technology

Positron emission computerized tomography (Positron Emission Tomograghy, PET) is at present in monitored in vivo The optimal images equipment of tumorigenesis process, it is possible to achieve to cell metabolism and the high-resolution image of function, from point Sub- level carries out noninvasive, three-dimensional, dynamic studies to the physiology of human body, biochemical process, and PET can be applied to the diagnosis of tumour, good evil Property differentiate, malignant tumour by stages, parting and tumor recurrence, the early diagnosis and discriminating of transfer, the selection of therapeutic scheme and chemotherapy, The detection of radiotherapeutic effect and the observation of tumour change procedure and the monitoring of rear situation.PET check dependent on broad spectrum activity or Specific targeting pet imaging agent is in target organ specific metabolic, absorption.¹⁸F-FDG is the most frequently used imagings of current PET Agent, but¹⁸F-FDG exist specificity it is poor, the shortcomings of occur false positive sometimes, cannot distinguish between tumour and inflammation, and some devices The basic metabolism of official's (brain, liver, heart) is originally just higher, in the diagnosis and discriminating of some brain tumors, lung cancer, prostate cancer etc. It is middle to there are many difficulties.Therefore¹⁸F-FDG is a kind of non-specific tumor developer, and diagnosis and research on tumour are also needed to New, the specific developer of research and development.

With the development of molecular biology, there is new understanding to the generation of cancer cell, development, migration from molecular level, Targeted drug, which accumulates, educates and gives birth to.The targeting small molecule tyrosine kinase inhibitors of EGF-R ELISA (EGFR) are based in recent years Research is very active, has had a variety of TKI micromoleculars medicines to be used for the treatment of cancer, wherein Erlotinib at present (Erlotinib) it is that effect preferably uses one of most wide biological targeting medicine.EGFR is a very attractive therapeutic target Point, it is in induced cell proliferation, invasion and attack, transfer and suppresses to play an important roll in the complicated signal path such as apoptosis.EGFR mistakes The tumour cell of degree expression receives some gene expressions in cell growth signal, active cell, accelerates cell differentiation, discharges blood Pipe generates the factor and promotees transfer factor, ultimately results in tumour and develops.EGFR families are made up of four transmembrane receptors, including EGFR (HERI/erbB-1), HER2 (erbB-2/neu), (HERI/erbB-3) and (HERI/erbB-4).Its protein knot Structure is made up of extracellular region, transmembrane region and intracellular region.Extracellular region is ligand binding domain, and transmembrane region is an independent α spiral, intracellular Area includes the carboxyl terminal of EGFR-TK (TK) area and several Tyr phosphorylation sites.

Radiolabeled tyrosine kinase inhibitor (TKI) turns into focus as the research of neoplasm tracing agent in recent years, It is numerous especially as the pet imaging agent researcher of PET/CT noninvasive tests.Mainly there are two major classes：¹⁸F mark and¹¹C flag Pet imaging agent.After Peter play-by-play in 1998¹¹After C-PD153035 several labeling methods, numerous positive electricity daughter nucleus The TKI micromoleculars tracer of element mark is reported by foreign study person.¹¹C flag has：¹¹C-Erlotinib、¹¹C- Gefitinib、¹¹C-M03、¹¹C-AZD8931、¹¹C-Vandetanib、¹¹C-Sorafenib etc..¹⁸What F was marked has：¹⁸F- Gefitinib、¹⁸F-Lapatinib、¹⁸F-ML04 etc..They are the Erlotinib classes with 4- amido quinazoline structures mostly Derivative.External Preliminary Results find that this micromolecular pet imaging agent can be combined with EGFR-TK, can reached The purpose of rii receptor, the positive tumor patient malignancy of EGFR imagings is high, easily shifts, high recurrence rate, in advance It is poor afterwards.The clinical targeted therapy of small molecule tyrosine kinase inhibitors biological targeting medicine is being instructed to have certain value simultaneously. Domestic scholars have also been made correlative study to such EGF-R ELISA pet imaging agent.Shandong Tumor Hospital is bright in gold Academician team pair¹¹C-PD153035 is furtherd investigate, it is found that its image results is proportionate with activity of EGFR, and to clinic Direction of medication usage is significant.The colleges and universities such as Harbin Medical University, Beijing Normal University, Zhejiang University are all to radioactivity in recent years The TKI class pet imaging agents of mark have done correlative study, have invented several new 4- amino-quinazoline pet imaging agents And patent protection.It can be seen that, such pet imaging agent has larger application prospect and market, and has part work It is confirmed by clinical practice.

Erlotinib extensive uses in clinic, its anticancer therapeutic is widely recognized, simultaneously because its own chemistry knot Structure feature, with preferable water solubility, in numerous radiolabeled TKI classes pet imaging agents,¹¹C-Erlotinib has Its unique advantage, can quickly be absorbed by internal organs and target target area through blood, be metabolized by liver sausage and urinary system.Memon 09 Year reports in Cancer Research first¹¹C-Erlotinib is as pet imaging agent in mice lung cancer transplantation tumor Imaging, research application EGFR expresses different three kinds of tumour cells HCC827, and NCI358, A549 is modeled, as a result shown respectively It is absorbed is expressed as positive correlation with EGFR.Its team is applied to clinic afterwards, has carried out Patients with Non-small-cell Lung imaging Research, it is found that its focal part has higher target/non-target ratio, can image some¹⁸The focus that F-FDG is not imaged is right¹⁸F-FDG rises Good supplement imaging effect has been arrived, has had very big guidance to anticipate the clinical personalized treatment of Erlotinibb biological targeting medicines Justice.Petrulli of Yale University in 2013 etc. is also reported¹¹EGFR expressions are being evaluated in C-ErlotinibPET/CT imagings With the outstanding behaviours of clinical guidance personalized treatment.Bahce etc. also studied 5 Exon deletions of EGFR 19 and not lacked with 5 Patients with Non-small-cell Lung clinic¹¹C-erlotinib is imaged, it is found that missing person can specificity intake¹¹C-Erlotinib, This can instruct the clinical targeting medication of Erlotinib.

Although scholars use in recent years¹¹C-Erlotinib is ground on basis and clinic study carefully in obtain many achievements, but also deposit It is limited in some shortcomings further to apply：(1)¹¹C half-life shorts, are difficult to make big sample research.(2)¹¹C-Erlotinib Can only there are the PET/CT center applications of accelerator, and the limited (agent produced every time of patient populations that single production can be met Amount is at most only capable of meeting 3 patients).(3) it is long in the case where reaching identical clinical effectiveness from health economics angle Half-life period nucleic is more cost-effective compared with the developer that Short half-life nuclides are marked.Consider from clinical practice situation, compel to be essential Develop half-life period longer positron radionuclide to be marked,¹⁸The Erlotinib of F marks is preferably to select, and it is hopeful into To be widely used in the pet imaging agent for instructing clinical personalized treatment in future clinical, and it is expected to do in biological medicine industry Go out certain contribution.The benzene ring position replaced by " click chemistry " method in Erlotinib 4- amino in our early-stage Studies It is upper to carry out¹⁸F is marked, and has applied for patent protection.We are attempted in Erlotinib 4- amido quinazoline structures in this research 6 or 7 at carry out¹⁸F is marked, to there is preferably targeting PET imaging results.

The content of the invention

It is an object of the invention to provide one kind¹⁸The EGFR positive electron tracers of F marks, structure such as formula A.

It is a further object of the present invention to provide this¹⁸The preparation method of the EGFR positive electron tracers of F marks, passes through a lower section Method one or method two are realized：

Method one, two-step method synthesis：Eluted with 1mL leacheates¹⁸QMA posts captured F, collect leacheate to reaction bulb In, 110 DEG C of heating is simultaneously constantly blown into high pure nitrogen, is azeotroped off H₂O.By acetonitrile solutions of the 1mL dissolved with 10mg intermediate precursors It is added to reaction bulb, 90 DEG C of reaction 15min；It is evaporated after above-mentioned reaction dissolvent, by dissolved with 1-3mg demethyls precursor and appropriate bases 0.3mL solution adds reaction bulb, 115 DEG C of reaction 10min.Cool down and the dilution of 10mL water is added after reaction solution, pass through Sep-Par C- 18 posts, and pillar is rinsed with 10mL water, C-18 posts are eluted with 2mL acetonitriles, leacheate is collected, the isolated mesh of HPLC is prepared through half Product is marked, mark top coal drawing is more than 99%.

Method two, one-step synthesis method：Eluted with 1mL leacheates¹⁸QMA posts captured F, collect leacheate to reaction bulb In, 110 DEG C of heating is simultaneously constantly blown into high pure nitrogen, is azeotroped off H₂O.0.3mL is added dissolved with the solution of 1mg one-step method precursors To reaction bulb, 120 DEG C of reaction 10min.Cool down and the dilution of 10mL water is added after reaction solution, by Sep-Par C-18 posts, be used in combination 10mL water rinses pillar, and C-18 posts are eluted with 2mL acetonitriles, collects leacheate, and the isolated target products of HPLC, mark are prepared through half Remember that top coal drawing is more than 99%.

Eluted in reaction¹⁸F is with the composition of 1mL solution：Oxygen -1,10- the phenodiazines of 12.0mg 4,7,13,16,21,24- six are double Ring [8.8.8] hexacosane is dissolved in 0.9mL acetonitriles plus 3.0mgK₂CO₃, or 4.3mg KHCO₃It is dissolved in 0.1mL water；First is gone in dissolving Based precursor and one-step method precursor solvent for use are anhydrous acetonitrile, anhydrous dimethyl sulphoxide (DMSO) or anhydrous N, N- dimethyl formyl One or more of mixing in amine (DMF).

In method one intermediate precursors be 1- methyl -4- [(4- aminomethyl phenyls) sulfonyloxy methoxysulfonyl] benzene, 1, One kind in the double toloxyl ethane of 2-, diethylene glycol double (tosylates), triethylene glycol double (tosylates)；Alkali used Species be：Sodium hydroxide, potassium hydroxide, hydrofining, sodium hydride, tetrabutylammonium, potassamide, Sodamide, butyl lithium, One kind in diisopropyl ammonia lithium, benzyl lithium, sodium methoxide, caustic alcohol；Wherein demethyl front body structure formula is as shown in formula C.

Front body structure formula is as shown in formula D in method two.

Third object of the present invention is to provide¹⁸The EGFR positive electrons tracer of F marks is used as the application of tumour track agent, reality Test and show¹⁸The EGFR positive electrons tracer of F marks can be used for the tumour of tumour especially EGF (EGFR) height expression PET is imaged, and is the new PET tumor imaging agent of a class.

The positive electron tracer of the present invention has good specificity, can positive identification skin factor acceptor (EGFR) high table Reach or the tumour that makes a variation.Its preparation method is convenient, simple, quick, can also pass through Radio-synthesis mould by synthetically prepared manually Block carries out Fully automated synthesis, sends special multifunction module and GE Tracerlab FX Fn modules through workflow reengineering by Beijing at present After can distinguish Fully automated synthesis, other brand synthesizers, which can also be realized, to be automatically synthesized, and can meet scientific research and clinical test need Ask.

Brief description of the drawings

Fig. 1 is intermediate 1-¹⁸F, 2- are to Methyl benzenesulfonyl epoxide ethane radio thin layer chromatogram (TLC) figure (solvent 100% ethyl acetate)；

Fig. 2 be [¹⁸F] -6-Fluoromethyl Erlotinib radio thin layers chromatogram (TLC) figure (second of solvent 100% Acetoacetic ester)；

Fig. 3 be [¹⁸F] -6-Fluoromethyl Erlotinib normal mice vivo biodistribution distribution maps.

Fig. 4 be HCC827 mice with tumor [¹⁸F] -6-Fluoromethyl Erlotinib Micro PET/CT imaging figures.

Embodiment

Below by experiment and the invention will be further described in conjunction with the embodiments, these embodiments are only used for illustration mesh , but do not limit the scope of the invention.In addition, embodiment 1-4 is in GE Tracerlab FX Fn Fully automated synthesis moulds It is automatically synthesized in block.

Embodiment 1

1) application medical cyclotron bombards¹⁸O water, passes through¹⁸O(pn)¹⁸F nuclear reactions production obtains 500mCi¹⁸F, and pass Lead in anion-exchange column, determine activity and with the 1mL mixed solutions (oxygen -1,10- of 12.0mg 4,7,13,16,21,24- six Diaza-bicyclo [8.8.8] hexacosane (K_2.2.2.) plus 3.0mgK₂CO₃It is dissolved in 0.1mL water and 0.9mL acetonitriles) will¹⁸F elution is to instead Answer in bottle；

2) azeotropic water removing at high-purity helium, 110 DEG C constantly is blown into reaction bulb, dried up；By 15mg precursors 1, (1,2- is double Toloxyl ethane) 1mL acetonitriles are dissolved in, add in reaction bulb, 90 DEG C of reaction 15min；80 DEG C are removed dry solvent after reaction terminates；

3) 2mg demethyl Erlotinib precursors 1 are dissolved in into 0.5mL DMSO, 5mgNaH to add in reaction bulb, 120 DEG C close Close reaction 10min；

4) dilution of 10mL water is added, C-18 posts are rinsed by Sep-Par Plus C-18 posts, then with 10mL water, after drying C-18 posts are eluted with 2mL acetonitriles, washing lotion is collected and is diluted with water to 5mL, the isolated target products of HPLC are prepared through half.

Through measuring and calculating, Radiochemical yield more than 20%, radiochemical purity is more than 99%.Fig. 1 is that embodiment 1 is prepared Intermediate 1-¹⁸F, 2- are to Methyl benzenesulfonyl epoxide ethane radio thin layer chromatogram (TLC) (ethyl acetate of solvent 100%)； Fig. 2 be embodiment 1 prepare [¹⁸F]-Erlotinib radio thin layers chromatogram (TLC) figure (acetic acid second of solvent 100% Ester).

Embodiment 2

2) azeotropic water removing at high-purity helium, 110 DEG C constantly is blown into reaction bulb, dried up；By the front body structure of method two such as Formula D (wherein R₁For CH₃-, R₂For TsOCH₂CH₂-) 1mg is dissolved in 0.3mLDMSO, add in reaction bulb, 120 DEG C of confined reactions 10min；

3) dilution of 10mL water is added, C-18 posts are rinsed by Sep-Par Plus C-18 posts, then with 10mL water, after drying C-18 posts are eluted with 2mL acetonitriles, washing lotion is collected and is diluted with water to 5mL, the isolated target products of HPLC are prepared through half.

Through measuring and calculating, Radiochemical yield more than 20%, radiochemical purity is more than 99%.

Embodiment 3

2) azeotropic water removing at high-purity helium, 110 DEG C constantly is blown into reaction bulb, dried up；By 10mg intermediate precursors 1- Methyl -4- [(4- aminomethyl phenyls) sulfonyloxy methoxysulfonyl] benzene is dissolved in 1mL acetonitriles, adds in reaction bulb, 90 DEG C of reactions 15min.80 DEG C are removed dry solvent after reaction terminates；

3) 3mg demethyls precursor 2 is dissolved in into 0.4mL DMF, 5mgKOH to add in reaction bulb, 120 DEG C of confined reactions 10min；

Embodiment 4

2) azeotropic water removing at high-purity helium, 110 DEG C constantly is blown into reaction bulb, dried up；By the precursor of method two, (structure is such as R in formula D₁For CH₃-, R₂For TsOCH₂CH₂OCH₂CH₂-) 1mg is dissolved in 0.3mLDMSO, add in reaction bulb, 120 DEG C of confined reactions 10min；

3) dilution of 10mL water is added, C-18 posts is rinsed by Sep-Par Plus C-18 posts, then with 10 water, is used after drying 2mL acetonitriles elute C-18 posts, collect washing lotion and are diluted with water to 5mL, the isolated target products of HPLC are prepared through half.Through surveying Calculate, Radiochemical yield more than 20%, radiochemical purity is more than 99%.

Embodiment 5 [¹⁸F] -6-Fluoromethyl Erlotinib internal stability experiment

Embodiment 1 prepare [¹⁸F] -6-Fluoromethyl Erlotinib are in the culture containing 10% hyclone In base 37 DEG C be incubated 4 hours after, radiochemical purity still reaches more than 97%, has good stability.

Embodiment 6 [¹⁸F] -6-Fluoromethyl Erlotinib internal distribution experiment

Embodiment 2 prepare [¹⁸F] -6-Fluoromethyl Erlotinib, in normal Kunming mouse vivo biodistribution point Butut such as Fig. 3, display medicine is mainly metabolized through liver sausage, and kidney also has a part metabolism, and blood removing speed is fast, and radioactivity is not in bone Height, medicine not defluorinate in vivo, stability is good.

The mice with tumor of embodiment 7 [¹⁸F] -6-Fluoromethyl Erlotinib Micro PET imagings

From EGFR height expression non-small cell lung cancer HCC827 lotus knurl mouse models, using embodiment 2 prepare [¹⁸F]- 6-Fluoromethyl Erlotinib carry out Micro PET imagings (Fig. 4), and Fig. 4 shows that tumor locus radioactive uptake is obvious (tissue) dirty higher than devices such as muscle, bone, lung, brains.

The preferred embodiment of the present invention is the foregoing is only, is only had for the purpose of the present invention illustrative but non-limiting；It is related Field those of ordinary skill to it in the spirit and scope that the claims in the present invention are limited it is understood that can carry out many modifications Or equivalent change, but fall within protection scope of the present invention.

Claims

1. it is a kind of¹⁸The EGFR positive electron tracers of F marks, it is characterised in that described positive electron tracer has shown in formula A Chemical constitution.

2. the preparation method of compound according to claim 1, it is characterised in that comprise the following steps：Pass through radioactivity Intermediate is carried out¹⁸The step marks of F two, the intermediate structure is shown below：

3. the preparation method of compound according to claim 2, it is characterised in that comprise the following steps：Use 1mL leacheates Elution¹⁸QMA posts captured F, collect leacheate into reaction bulb, and 110 DEG C of heating is simultaneously constantly blown into high pure nitrogen, is azeotroped off H₂O；1mL is added to reaction bulb, 90 DEG C of reaction 15min dissolved with the acetonitrile solution of 10mg intermediate precursors；It is evaporated above-mentioned reaction After solvent, reaction bulb, 115 DEG C of reaction 10min will be added dissolved with the 0.3mL solution of 1-3mg demethyls precursor and appropriate bases；Cooling The dilution of 10mL water is added after reaction solution, pillar is rinsed by Sep-Par C-18 posts, and with 10mL water, C- is eluted with 2mL acetonitriles 18 posts, collect leacheate, and the isolated target products of HPLC are prepared through half, and mark top coal drawing is more than 99%.

4. the preparation method of compound according to claim 2, it is characterised in that comprise the following steps：Use 1mL leacheates Elution¹⁸QMA posts captured F, collect leacheate into reaction bulb, and 110 DEG C of heating is simultaneously constantly blown into high pure nitrogen, is azeotroped off H₂O；0.3mL is added to reaction bulb, 120 DEG C of reaction 10min dissolved with the solution of 1mg one-step method precursors；Cool down and add after reaction solution Enter the dilution of 10mL water, rinse pillar by Sep-Par C-18 posts, and with 10mL water, C-18 posts are eluted with 2mL acetonitriles, collect and drench Washing lotion, the isolated target products of HPLC are prepared through half, and mark top coal drawing is more than 99%.

5. the preparation method of the compound according to claim 3 or 4, it is characterised in that the elution¹⁸QMA captured F The solution of post includes：12.0mg 4,7,13,16,21,24- six oxygen -1,10- diaza-bicyclos [8.8.8] hexacosane is dissolved in 0.9mL acetonitriles, 3.0mgK₂CO₃Or 4.3mg KHCO₃It is dissolved in 0.1mL water；Dissolve used in demethyl precursor and one-step method precursor Solvent is one or more of mixed in anhydrous acetonitrile, anhydrous dimethyl sulphoxide (DMSO) or anhydrous N,N-dimethylformamide (DMF) Close.

6. the preparation method of compound according to claim 3, it is characterised in that the intermediate precursors are to be selected from：1- The double toloxyl ethane of methyl -4- [(4- aminomethyl phenyls) sulfonyloxy methoxysulfonyl] benzene, 1,2-, the double (first of diethylene glycol Benzene sulfonate), one kind in triethylene glycol double (tosylate).

7. the preparation method of compound according to claim 3, it is characterised in that the alkali is to be selected from：Sodium hydroxide, hydrogen Potassium oxide, hydrofining, sodium hydride, tetrabutylammonium, potassamide, Sodamide, butyl lithium, diisopropyl ammonia lithium, benzyl lithium, One kind in sodium methoxide, caustic alcohol.

8. the preparation method of compound according to claim 3, it is characterised in that the structural formula of the demethyl precursor is such as Shown in following formula：

9. method according to claim 4, it is characterised in that shown in the following D of structural formula of the one-step method precursor：

10. EGFR positive electrons tracer is used for the application for preparing PET tumor imaging agent according to claim 1.