CN107522673B - 1,2,4, 5-tetrazine compound for bioorthogonal reaction and preparation method and application thereof - Google Patents
1,2,4, 5-tetrazine compound for bioorthogonal reaction and preparation method and application thereof Download PDFInfo
- Publication number
- CN107522673B CN107522673B CN201710702980.3A CN201710702980A CN107522673B CN 107522673 B CN107522673 B CN 107522673B CN 201710702980 A CN201710702980 A CN 201710702980A CN 107522673 B CN107522673 B CN 107522673B
- Authority
- CN
- China
- Prior art keywords
- compound
- reaction
- bioorthogonal
- follows
- tetrazine
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/08—Six-membered rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/002—Heterocyclic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F13/00—Compounds containing elements of Groups 7 or 17 of the Periodic System
- C07F13/005—Compounds without a metal-carbon linkage
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides a novel 1,2,4, 5-tetrazine compound for bioorthogonal reaction, a preparation method and application thereof. The invention also provides a new structure99mTc/Re labeled 1,2,4, 5-tetrazine compound and its preparation method, and its application in imaging based on bioorthogonal reaction, especially in brain imaging. The structure of the labeled compound is shown as the formula (I):
the reaction kinetics experiment shows that the molecule has high reaction activity with trans-cyclooctene derivative, and the normal mouse in vivo biodistribution experiment shows that99mThe Tc-labeled molecules can effectively pass through a blood brain barrier, and the brain clearance is fast, so that the Tc-labeled molecules can become a novel imaging agent for bioorthogonal reaction in the brain.
Description
Technical Field
The invention relates to the technical field of radiopharmaceutical chemistry and clinical nuclear medicine, in particular to a 1,2,4, 5-tetrazine compound for bioorthogonal reaction and a preparation method and application thereof.
Background
Bioorthogonal reactions (bioorthogonal reactions) are being widely used in the fields of biology, chemistry, etc., and these reactions can specifically occur under physiological conditions in vivo, do not interfere with other biochemical reactions occurring simultaneously in vivo, and do not cause damage to the organism and target biomolecules. The ideal bio-orthogonal reaction needs to satisfy the following conditions: (1) selectivity with respect to other potentially active functional groups present on the biomolecule; (2) in an aqueous medium; (3) under physiological pH conditions; (4) the use of a low concentration of reactants has a faster reaction rate at normal temperature. However, most of the existing bio-orthogonal reactions are not able to satisfy all these conditions at the same time, and the specific labeling of biomolecules in biological environments by chemical reactions remains very challenging.
The research application of the monovalent copper catalyzed azide-alkyne [3+2] cycloaddition (Cu (I) -catalyzed [3+2] azido-alkylsynthesis, CuAAC) reaction and the azide-alkyne [3+2] cycloaddition (SPAAC) reaction promoted by ring tension as the traditional bioorthogonal reaction is limited by the lower reaction rate. The recently emerging anti-electron demand Diels-Alder (IEDDA) reaction has received much attention because of its incomparable reaction kinetics, extremely high reaction orthogonality, and biocompatibility. Among them, the research on the IEDDA reaction between dienophiles such as 1,2,4, 5-tetrazine and trans-cyclooctene is the most extensive, and the reaction meets the requirements of biological applications, namely, high selectivity, mild reaction conditions of aqueous phase and high reaction speed, so that the reaction can be efficiently carried out under the low concentration condition close to the protein concentration level in vivo, and even can be used for researching the cell function and the kinetic process. The IEDDA reaction has great application potential in basic biology, molecular imaging, disease treatment and other aspects.
The excellent kinetic properties make it possible to radiolabel the IEDDA reaction in vivo at low concentrations by a pretargeting method. Under in vivo conditions, very low concentrations of radiolabeled compounds can be efficiently attached to target molecules in a relatively short time by the IEDDA reaction. Studies have shown that radiolabeled trans-cyclooctene and its derivatives are metabolized too rapidly in vivo, compared to the more practical use of radiolabels of 1,2,4, 5-tetrazine compounds. The earliest radiolabeled 1,2,4, 5-tetrazine compounds were mostly longer half-life radiometals (e.g., Galvanic elements)64Cu，177Lu，111In, etc.) complexes later extended to covalently linked short half-life nuclides18F and11c, etc. to99mTc-labelled 1,2,4, 5-tetrazine compounds have been poorly studied, and99mtc nuclide has proper half-life, moderate ray energy and rich coordination chemistry, and the matched SPECT imaging is more popular and economical, so the development of the Tc nuclide is realized99mThe Tc marked 1,2,4, 5-tetrazine compound has good application prospect. In addition, from the perspective of targeted area analysis, the current related research mainly focuses on imaging outside the central nervous system such as tumor, and there is only a few application designs for imaging related targets in the brain, and there is no research on the property of 1,2,4, 5-tetrazine compound crossing the blood brain barrier.
Disclosure of Invention
The invention aims to provide a novel 1,2,4, 5-tetrazine compound for bioorthogonal reaction, and a preparation method and application thereof.
In order to achieve the object of the present invention, the present invention provides a 1,2,4, 5-tetrazine compound for bioorthogonal reaction, which has the structure represented by formula (IV):
wherein X is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2Ph is phenyl and Tr is trityl.
The compound of formula (IV) can be prepared as follows:
1) weighing 5mmol of HO-X-CN, 25mmol of acetonitrile and 2mmol of anhydrous nickel chloride, dropwise adding 125mmol of 80% hydrazine hydrate solution into a 250mL round-bottom flask, heating at 60 ℃ for reaction for 24 hours, cooling to normal temperature, weighing 72.5mmol of sodium nitrite, adding water for dissolving, dropwise adding, then placing into an ice water bath, dropwise adding 2M hydrochloric acid until no bubbles are generated, extracting with ethyl acetate, combining organic phases, drying with anhydrous sodium sulfate, performing rotary evaporation concentration, and separating with a silica gel column to obtain a compound (II);
2) weighing 1mmol of compound (II) and 1.5mmol of p-toluenesulfonyl chloride, adding 10mL of anhydrous dichloromethane and 1mmol of triethylamine into a 100mL round-bottom flask, stirring at room temperature for 12 hours, concentrating by rotary evaporation, and separating by a silica gel column to obtain a compound (III);
3) weighing 0.2mmol of BAT-Boc (N-tert-butyloxycarbonyl-S, S' -trityl-bis (2-mercaptoethyl) ethylenediamine), 0.3mmol of compound (III) and 0.4mmol of N, N-diisopropylethylamine in a 50mL round-bottom flask, adding acetonitrile to dissolve, heating at 60 ℃ for reaction for 48 hours, carrying out rotary evaporation and concentration, and separating by using a silica gel column to obtain a compound of a formula (IV);
wherein the structures of the compound (II), the compound (III) and the BAT-Boc are respectively as follows:
x is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2Ph is phenyl, Tr is trityl, and OTs is p-toluenesulfonyl.
The invention also provides the application of the compound shown in the formula (IV) in the preparation of an imaging agent for bioorthogonal reaction, in particular to the imaging agent for bioorthogonal reaction in brain.
The invention also provides an imaging agent for bioorthogonal reaction, which has a structure shown in the formula (I):
wherein X is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2-, Ph is phenyl; m is Re or99mTc。
The developing agent of the invention can be prepared according to the following method:
when M is Re, the compound is prepared as follows: placing 0.1mmol compound (IV) in 50mL round-bottom flask, placing in ice water bath, adding 2mL trifluoroacetic acid, stirring for 5min, and adding40 μ L of triethylsilane, stirred for 10min, trifluoroacetic acid removed under reduced pressure, and 0.1mmol (PPh) of the resulting mixture was added3)2ReOCl320mL of dichloromethane: heating and refluxing a mixed solvent of 9:1 (volume ratio) methanol and a proper amount of anhydrous sodium acetate at 90 ℃ for 2 h; after TLC monitoring reaction, decompression removing solvent, silica gel column chromatography separation purification to obtain.
When M is99mAt Tc, the compound is prepared as follows: 0.05mg of Compound (IV) was dissolved in 100. mu.L of trifluoroacetic acid and allowed to stand at room temperature for 5 min. Adding 2 mu L of triethylsilane, standing at room temperature for 10min, and drying with nitrogen; dissolving in 50 μ L of anhydrous ethanol, and adding the newly prepared99mTc-sodium glucoheptonate (1-100mCi, 0.5-2.0mL) is heated at 90 ℃ for 10min to obtain the product.
The invention also provides application of the imaging agent in the field of molecular imaging based on bioorthogonal reaction.
The invention also provides application of the imaging agent in the field of molecular imaging based on bioorthogonal reaction in brain.
The invention further provides application of the imaging agent in single photon tomography imaging.
The invention has the following advantages:
the invention provides a 1,2,4, 5-tetrazine compound with a novel structure and application in bioorthogonal reaction99mTc/Re marked 1,2,4, 5-tetrazine compound, reaction kinetics experiment shows that the molecule has high reaction activity with trans-cyclooctene compound, and normal mouse in vivo biodistribution experiment shows99mThe Tc-labeled molecules are all able to pass effectively through the blood-brain barrier, in particular99mTc]15 (corresponding to the compound of example 19) is highest in initial intracerebral uptake at 2min and brain clearance is also faster, thus being a novel imaging agent for bioorthogonal reactions in the brain.
Drawings
FIG. 1 is a scheme showing the synthesis of 1,2,4, 5-tetrazine compounds in examples 13 to 16 of the present invention.
FIG. 2 is a drawing showing the preparation of 1,2,4, 5-tetrazine compounds in examples 17 to 20 of the present invention99mTc marks the route.
FIG. 3 is a graph showing the reaction kinetics of 1,2,4, 5-tetrazine compounds in examples 13 to 16 of the present invention; wherein A-D correspond to rhenium complexes 13-16, respectively.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 Synthesis of intermediate 1
665.8mg of 4- (hydroxymethyl) benzonitrile, 1026.3mg of acetonitrile and 259.2mg of anhydrous nickel chloride are weighed into a 250mL round-bottom flask, 7821.9mg of hydrazine hydrate (concentration: 80%) is added dropwise, the reaction is heated at 60 ℃ for 24 hours, the reaction is cooled to normal temperature, and 5002.5mg of sodium nitrite is weighed, dissolved in water and added dropwise. Then placing the mixture in an ice water bath, dropwise adding 2M hydrochloric acid until no bubbles are generated, extracting with ethyl acetate, combining organic phases, drying with anhydrous sodium sulfate, performing rotary evaporation concentration, and separating with a silica gel column to obtain a product, namely an intermediate 1, wherein the structure is as follows, and the yield is as follows: 44 percent.1H NMR(400MHz,CDCl3)δ8.58(d,J＝7.9Hz,2H),7.59(d,J＝8.0Hz,2H),4.84(s,2H),3.10(s,3H).
Example 2 Synthesis of intermediate 2
The intermediate 2 is prepared by the reaction of 3- (2-hydroxyethoxy) propionitrile, acetonitrile and hydrazine hydrate, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the preparation of the intermediate 1, the structure is as follows, and the yield is as follows: 17 percent.1H NMR(400MHz,CDCl3)δ4.11(t,J＝6.2Hz,2H),3.69–3.67(m,2H),3.63–3.60(m,2H),3.58(t,J＝6.2Hz,2H),3.05(s,3H),2.56(s,1H).
Example 3 Synthesis of intermediate 3
Prepared by the reaction of 4- (2-hydroxyethoxy) benzonitrile with acetonitrile and hydrazine hydrateThe intermediate 3 is obtained, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the preparation of the intermediate 1, the structure is as follows, and the yield is as follows: 44 percent.1H NMR(400MHz,CDCl3)δ8.55(d,J＝8.9Hz,2H),7.10(d,J＝8.9Hz,2H),4.20(t,J＝4.6Hz,2H),4.03(t,J＝4.6Hz,2H),3.06(s,3H).
Example 4 Synthesis of intermediate 4
The intermediate 4 is prepared by the reaction of 4- (3-hydroxypropyl) benzonitrile with acetonitrile and hydrazine hydrate, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the preparation of the intermediate 1, the structure is as follows, and the yield is as follows: 32 percent.1H NMR(400MHz,CDCl3)δ8.51(d,J＝8.3Hz,2H),7.43(d,J＝8.3Hz,2H),3.72(t,J＝6.4Hz,2H),3.08(s,3H),2.83(t,J＝7.9Hz,2H),2.01–1.92(m,2H).
Example 5 Synthesis of intermediate 5
298.6mg of intermediate 1, 423.2mg of p-toluenesulfonyl chloride were weighed into a 100mL round-bottomed flask, and about 10mL of anhydrous dichloromethane and 154.7mg of triethylamine were added to stir the reaction at room temperature for 12 hours. Rotary evaporation and concentration, and silica gel column separation to obtain the product with the following structure and yield: 14 percent.1H NMR(400MHz,CDCl3)δ8.55(d,J＝8.2Hz,2H),7.83(d,J＝8.1Hz,2H),7.48(d,J＝8.2Hz,2H),7.35(d,J＝8.1Hz,2H),5.16(s,2H),3.10(s,3H),2.45(s,3H).
Example 6 Synthesis of intermediate 6
The intermediate 6 is prepared by the reaction of the intermediate 2 and the paratoluensulfonyl chloride, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the preparation of the intermediate 5, the structure is as follows, and the yield is as follows: 35 percent.1H NMR(400MHz,CDCl3)δ7.75(d,J＝8.3Hz,2H),7.34(d,J＝8.0Hz,2H),4.10–4.06(m,2H),4.03(t,J＝6.3Hz,2H),3.68–3.63(m,2H),3.50(t,J＝6.3Hz,2H),3.05(s,3H),2.45(s,3H).
Example 7 Synthesis of intermediate 7
The intermediate 7 is prepared by the reaction of the intermediate 3 and p-toluenesulfonyl chloride, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the intermediate 5, the structure is as follows, and the yield is as follows: 67%.1H NMR(400MHz,CDCl3)δ8.51(d,J＝8.9Hz,2H),7.83(d,J＝8.3Hz,2H),7.35(d,J＝8.1Hz,2H),6.96(d,J＝8.9Hz,2H),4.43(t,J＝5.0Hz,2H),4.26(t,J＝5.0Hz,2H),3.06(s,3H),2.45(s,3H).
EXAMPLE 8 Synthesis of intermediate 8
The intermediate 8 is prepared by the reaction of the intermediate 4 and p-toluenesulfonyl chloride, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the preparation of the intermediate 5, the structure is as follows, and the yield is as follows: 26 percent.1H NMR(400MHz,CDCl3)δ8.45(d,J＝8.3Hz,2H),7.80(d,J＝8.3Hz,2H),7.35(d,J＝8.1Hz,2H),7.29(d,J＝8.3Hz,2H),4.06(t,J＝6.1Hz,2H),3.09(s,3H),2.77(t,J＝7.5Hz,2H),2.46(s,3H),2.07–1.99(m,2H).
Example 9 Synthesis of a Label precursor 9
152.9mg of BAT-Boc,106.9mg of intermediate 5 and 51.6mg of N, N-diisopropylethylamine were weighed into a 50mL round-bottomed flask, dissolved by adding acetonitrile, and reacted at 60 ℃ for 48 hours. Rotary evaporation and concentration, and silica gel column separation to obtain the product with the following structure and yield: 76 percent.1H NMR(400MHz,CDCl3)δ8.52–8.43(m,2H),7.40–7.35(m,14H),7.26–7.20(m,12H),7.20–7.14(m,6H),3.50–3.40(m,2H),3.09(s,3H),3.03–2.84(m,4H),2.45–2.38(m,2H),2.35–2.22(m,6H),1.36–1.29(m,9H).
Example 10 Synthesis of a Mark precursor 10
The intermediate 6 and BAT-Boc were reacted to produce the labeled precursor 10, which was prepared in the same proportions of starting materials, solvents, reaction conditions, etc. as the intermediate 9, and was structured as follows, in terms of yield: 36 percent.1H NMR(400MHz,CDCl3)δ7.42–7.36(m,12H),7.29–7.22(m,12H),7.21–7.14(m,6H),3.95(t,J＝6.5Hz,2H),3.50(t,J＝6.5Hz,2H),3.32(t,J＝6.1Hz,2H),3.00(s,3H),2.97–2.73(m,4H),2.41–2.14(m,10H),1.36(s,9H).
Example 11 Synthesis of a Label precursor 11
The intermediate 7 and BAT-Boc were reacted to produce the labeled precursor 11, which was prepared in the same proportions of starting materials, solvents, reaction conditions, etc. as the intermediate 9, and was structured as follows, in terms of yield: 42 percent.1H NMR(400MHz,CDCl3)δ8.50(d,J＝8.6Hz,2H),7.44–7.35(m,12H),7.30–7.22(m,12H),7.21–7.14(m,6H),6.99(d,J＝8.9Hz,2H),3.90(s,2H),3.46–3.41(m,2H),3.06(s,3H),3.03–2.85(m,4H),2.51–2.24(m,8H),1.37(s,9H).
Example 12 Synthesis of a Label precursor 12
The intermediate 8 and BAT-Boc were reacted to produce the labeled precursor 12, which was prepared in the same proportions of starting materials, solvents, reaction conditions, etc. as the intermediate 9, and was structured as follows, in terms of yield: 44 percent.1H NMR(400MHz,CDCl3)δ8.45(d,J＝8.3Hz,2H),7.40–7.37(m,12H),7.32(d,J＝8.4Hz,2H),7.27–7.22(m,12H),7.19–7.14(m,6H),3.08(s,3H),3.05–2.81(m,4H),2.60(t,J＝7.7Hz,2H),2.38–2.19(m,10H),1.64–1.57(m,2H),1.36(s,9H).
EXAMPLE 13 Synthesis of rhenium Complex 13
Weighing 94.9mg of labeled precursor 9 into a 50mL round bottom flask, placing in an ice-water bath, adding 2mL of trifluoroacetic acid, stirring for 5min, adding 40. mu.L of triethylsilane, stirring for 10min, removing trifluoroacetic acid under reduced pressure, adding 83.3mg (PPh)3)2ReOCl320mL of dichloromethane: a mixed solvent of methanol 9:1(v: v) and a proper amount of anhydrous sodium acetate, and heating and refluxing for 2h at 90 ℃. After TLC monitoring reaction, decompression removes solvent, silica gel column chromatography separation purification to obtain the product, the structure is as follows, yield: 11 percent.1HNMR(400MHz,CDCl3)δ8.70(d,J＝7.9Hz,2H),7.70(d,J＝8.0Hz,2H),5.27(d,J＝14.4Hz,1H),4.85(d,J＝14.2Hz,1H),4.27–4.07(m,3H),3.91–3.81(m,1H),3.78–3.68(m,1H),3.46–3.35(m,1H),3.14(s,3H),3.23–3.02(m,3H),2.93–2.80(m,2H),1.80–1.69(m,1H).
EXAMPLE 14 Synthesis of rhenium Complex 14
From the labeling precursors 10 and (PPh)3)2ReOCl3The rhenium complex 14 is prepared by reaction, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the rhenium complex 13, the structure is as follows, and the yield is as follows: 15 percent.1H NMR(400MHz,CDCl3)δ4.20–4.01(m,5H),3.92–3.73(m,5H),3.60(t,J＝6.1Hz,2H),3.40–3.25(m,3H),3.20–3.12(m,1H),3.06(s,3H),3.03–2.96(m,2H),2.66–2.58(m,1H),1.71–1.64(m,1H).
EXAMPLE 15 Synthesis of rhenium Complex 15
From the labeling precursors 11 and (PPh)3)2ReOCl3The rhenium complex 15 is prepared by reaction, the proportion of raw materials, solvent, reaction conditions and the like of the reaction are the same as those of the rhenium complex 13, the structure is as follows, and the yield is as follows: 44 percent.1H NMR(400MHz,CDCl3)δ8.59(d,J＝8.9Hz,2H),7.12(d,J＝9.0Hz,2H),4.58–4.44(m,3H),4.20–4.03(m,4H),3.85–3.81(m,1H),3.61–3.50(m,2H),3.43–3.36(m,1H),3.34–3.26(m,1H),3.22–3.17(m,1H),3.08(s,3H),3.07–3.00(m,1H),2.84–2.79(m,1H),1.93–1.86(m,1H).
EXAMPLE 16 Synthesis of rhenium Complex 16
From the labeling precursors 12 and (PPh)3)2ReOCl3The rhenium complex 16 is prepared by reaction, the raw material proportion, the solvent, the reaction conditions and the like of the reaction are the same as those of the rhenium complex 13, the structure is as follows, and the yield is as follows: 27 percent.1H NMR(400MHz,CDCl3)δ8.55(d,J＝8.4Hz,2H),7.43(d,J＝8.4Hz,2H),4.14–4.04(m,3H),3.92–3.84(m,1H),3.80–3.75(m,1H),3.71–3.63(m,1H),3.38–3.24(m,3H),3.22–3.14(m,1H),3.09(s,3H),3.06–3.00(m,1H),2.99–2.93(m,1H),2.85–2.77(m,2H),2.76–2.70(m,1H),2.26–2.13(m,2H),1.79–1.68(m,1H).
The synthetic routes of the 1,2,4, 5-tetrazine compounds of examples 13-16 are shown in FIG. 1.
99mTc labelled compounds may be prepared by conventional methods known in the art, the general labelling reaction being shown in FIG. 2, as follows: 0.05mg of the labeled precursor was dissolved in 100. mu.L of trifluoroacetic acid and allowed to stand at room temperature for 5 min. Adding 2 mu L of triethylsilane, standing at room temperature for 10min, and drying by nitrogen. Dissolving with 50 μ L of anhydrous ethanol, and adding freshly prepared99mHeating Tc-sodium glucoheptonate at 90 deg.c for 10min to obtain the product99mTc labels the complex.
The resulting complex was subjected to HPLC detection. HPLC fractionationThe analysis conditions are as follows: venusil MP C18 column (Agela technologies,5 μm, 4.6X 250 mm); CH (CH)3CN/H2O is 80%/20%; flow rate, 1 mL/min.
Prepared according to the above method99mTc-labelled complexes are shown in Table 1.
TABLE 199mTc-labelled Complex (examples 17 to 20)
Example 211 Effect test of 2,4, 5-Tetrazine Compound
The bioorthogonal reactivity of the compound of the present invention was evaluated by a reaction kinetics experiment, and the initial brain uptake and brain clearance properties of the labeled compound were evaluated by a normal mouse in vivo distribution experiment.
1. Experiment of reaction kinetics
Reacting 1,2,4, 5-tetrazine compound with trans-cyclooctene derivative (structure as below) under quasi-first-order reaction condition, monitoring reaction progress by ultraviolet-visible spectrum to determine reaction rate constant, and quantitatively
The bio-orthogonal reactivity of 1,2,4, 5-tetrazine compounds was evaluated.
1.1 Experimental procedure:
respectively preparing a 1,2,4, 5-tetrazine rhenium complex solution with the concentration of 5 mu M and a trans-cyclooctene compound solution with the concentration of 50 mu M (both are 10% ethanol-containing aqueous solutions, and for the rhenium complex 14, the concentrations of the two solutions are respectively 50 mu M and 500 mu M), respectively taking 1mL of each solution, adding the two solutions into a cuvette, quickly mixing the two solutions, collecting data by using a Kinetics program, wherein the monitoring wavelength is listed in Table 2, and the data collection interval is 1 s. Obtaining an apparent rate constant through software fitting, and further obtaining a second-order reaction rate constant k2. Each set of reactions was assayed in triplicate.
1.2 Experimental results:
two obtained by experimentRate constant k of order reaction2See table 2.
TABLE 2 Rate constants for the reaction of 1,2,4, 5-tetrazine compounds prepared in accordance with the invention with Trans-cyclooctene compounds
The experiments show that the compounds have high reactivity with trans-cyclooctene derivatives.
2. In vivo distribution experiment in Normal mice
Through in vivo distribution experiment research99mPharmacokinetic properties of Tc-labeled compounds in normal ICR mice.
2.1 Experimental procedures
A labeled compound (5-10 μ Ci) was injected into normal mice (ICR, male, 20-22g) (n ═ 5) from the tail vein (100 μ L of physiological saline solution, containing 10% ethanol), decapitated at 2 minutes, 10 minutes, 30 minutes, and 60 minutes after injection, respectively, and relevant organs were dissected out, and wet weight and radioactive counts were measured. Data are expressed as percent radioactivity in organs (% ID/organ) and percent radioactivity per gram of organs (% ID/g).
2.2 results of the experiment
The experimental results are shown in Table 3, and the invention provides99mThe Tc marker compounds are each capable of effectively passing through the blood-brain barrier, particularly [ 2]99mTc]15 at 2min initial brain uptake is highest and brain clearance is also faster, thus can be used as imaging agent for bioorthogonal reactions in the brain.
TABLE 399mNormal mouse (n-5) in vivo biodistribution of Tc-tagged compoundsa
aExpressed as% ID/g, mean. + -. standard deviation;bindicates% ID/organ.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1.1, 2,4, 5-tetrazine compounds for bioorthogonal reactions, characterized by the structural formula (IV):
wherein X is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2Ph is phenyl and Tr is trityl.
2. The method for preparing a 1,2,4, 5-tetrazine compound for bioorthogonal reaction of claim 1, comprising the steps of:
1) weighing 5mmol of HO-X-CN, 25mmol of acetonitrile and 2mmol of anhydrous nickel chloride, dropwise adding 125mmol of 80% hydrazine hydrate solution into a 250mL round-bottom flask, heating at 60 ℃ for reaction for 24 hours, cooling to normal temperature, weighing 72.5mmol of sodium nitrite, adding water for dissolving, dropwise adding, then placing into an ice water bath, dropwise adding 2M hydrochloric acid until no bubbles are generated, extracting with ethyl acetate, combining organic phases, drying with anhydrous sodium sulfate, performing rotary evaporation concentration, and separating with a silica gel column to obtain a compound (II);
2) weighing 1mmol of compound (II) and 1.5mmol of p-toluenesulfonyl chloride in a 100mL round-bottom flask, adding 10mL of anhydrous dichloromethane and 1mmol of triethylamine, stirring at room temperature for reaction for 12 hours, concentrating by rotary evaporation, and separating by a silica gel column to obtain a compound (III);
3) weighing 0.2mmol BAT-Boc, 0.3mmol compound (III) and 0.4mmol N, N-diisopropylethylamine in a 50mL round-bottom flask, adding acetonitrile to dissolve, heating at 60 ℃ for reaction for 48 hours, concentrating by rotary evaporation, and separating with silica gel column to obtain the compound of formula (IV);
wherein the structures of the compound (II), the compound (III) and the BAT-Boc are respectively as follows:
x is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2Ph is phenyl, Tr is trityl, and OTs is p-toluenesulfonyl.
3. Use of the 1,2,4, 5-tetrazine compound for bioorthogonal reactions as claimed in claim 1 for the preparation of an imaging agent useful for bioorthogonal reactions in the brain.
4. An imaging agent for bioorthogonal reactions, characterized in that it has the structure shown in formula (I):
wherein X is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2-, Ph is phenyl; m is Re or99mTc。
5. The method for preparing an imaging agent for bioorthogonal reaction according to claim 4, wherein when M is Re, the compound is prepared as follows: placing 0.1mmol of compound (IV) in 50mL round-bottom flask, placing in ice-water bath, adding 2mL trifluoroacetic acid, stirring for 5min, adding 40 μ L triethylsilane, stirring for 10min, removing trifluoroacetic acid under reduced pressure, adding 0.1mmol (PPh)3)2ReOCl320mL of dichloromethane: heating and refluxing a mixed solvent with methanol volume ratio of 9:1 and a proper amount of anhydrous sodium acetate at 90 ℃ for 2 h; after TLC monitoring reaction is finished, removing the solvent under reduced pressure, and separating and purifying by silica gel column chromatography to obtain the product;
wherein the structure of the compound (IV) is shown as the formula (IV):
wherein X is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2Ph is phenyl and Tr is trityl.
6. The method for preparing the imaging agent for bioorthogonal reaction of claim 4, wherein when M is99mAt Tc, the compound is prepared as follows: 0.05mg of Compound (IV) was dissolved in 100. mu.L of trifluoroacetic acid and allowed to stand at room temperature for 5 min. Adding 2 mu L of triethylsilane, standing at room temperature for 10min, and drying with nitrogen; dissolving in 50 μ L of anhydrous ethanol, and adding 1-100mCi99mHeating Tc-sodium glucoheptonate 0.5-2.0mL at 90 deg.c for 10min to obtain the product;
wherein the structure of the compound (IV) is shown as the formula (IV):
wherein X is-Ph-CH2-、-CH2CH2-O-CH2CH2-、-Ph-OCH2CH2-or-Ph-CH2CH2CH2Ph is phenyl and Tr is trityl.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710702980.3A CN107522673B (en) | 2017-08-16 | 2017-08-16 | 1,2,4, 5-tetrazine compound for bioorthogonal reaction and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710702980.3A CN107522673B (en) | 2017-08-16 | 2017-08-16 | 1,2,4, 5-tetrazine compound for bioorthogonal reaction and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107522673A CN107522673A (en) | 2017-12-29 |
| CN107522673B true CN107522673B (en) | 2020-05-19 |
Family
ID=60681162
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710702980.3A Active CN107522673B (en) | 2017-08-16 | 2017-08-16 | 1,2,4, 5-tetrazine compound for bioorthogonal reaction and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107522673B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11796610B2 (en) | 2018-03-09 | 2023-10-24 | Duke University | Compositions as molecular tags for hyperpolarization NMR and magnetic resonance and methods of making and using same |
| CN110496233B (en) * | 2019-09-05 | 2022-07-19 | 复旦大学附属中山医院 | SPECT imaging agent, marked precursor thereof, preparation method, composition and application thereof |
| CN110954514B (en) * | 2019-12-02 | 2022-06-21 | 上海交通大学 | Fluorescent tracing system and method suitable for researching in-vivo and in-vitro distribution of liposome |
| CN111138337B (en) * | 2019-12-26 | 2021-06-22 | 清华大学 | Bicyclo [4.1.0] heptane nitrosourea derivatives for bioorthogonal reaction and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103096936A (en) * | 2010-08-20 | 2013-05-08 | 通用电气健康护理有限公司 | Imaging tuberculosis with pyrazinamide contrast agents |
| WO2013152359A1 (en) * | 2012-04-06 | 2013-10-10 | The Regents Of The University Of California | Novel tetrazines and method of synthesizing the same |
| CN106496275A (en) * | 2016-09-07 | 2017-03-15 | 北京师范大学 | There is the N of high-affinity with A β plaque block2S22 aryl benzothiazole compound of class and preparation method and application |
-
2017
- 2017-08-16 CN CN201710702980.3A patent/CN107522673B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103096936A (en) * | 2010-08-20 | 2013-05-08 | 通用电气健康护理有限公司 | Imaging tuberculosis with pyrazinamide contrast agents |
| WO2013152359A1 (en) * | 2012-04-06 | 2013-10-10 | The Regents Of The University Of California | Novel tetrazines and method of synthesizing the same |
| CN106496275A (en) * | 2016-09-07 | 2017-03-15 | 北京师范大学 | There is the N of high-affinity with A β plaque block2S22 aryl benzothiazole compound of class and preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 99mTc 标记的苯并噻唑类 Aβ 斑块分子探针的合成与评价;张晓阳，等;《中国化学会第30届学术年会》;20160701 * |
| Efficient [18F]AlF Radiolabeling of ZHER3:8698 Affibody Molecule for Imaging of HER3 Positive Tumors;Chiara Da Pieve等;《Bioconjugate Chemistry》;20160629;第27卷;第1839页左栏第1段，第1840页方案1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107522673A (en) | 2017-12-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107522673B (en) | 1,2,4, 5-tetrazine compound for bioorthogonal reaction and preparation method and application thereof | |
| EP2070897B1 (en) | Method of rapid methylation, kit for preparing pet tracer and method of producing pet tracer | |
| CN110627868B (en) | A kind of18F-labeled compound and legumain-targeted PET imaging probe | |
| EP1926500A2 (en) | Methods of making radiolabeled tracers and precursors thereof | |
| CN112175025B (en) | Glucose derivative containing benzene ring and application thereof | |
| CN112209970B (en) | Preparation method and application of technetium-99 m labeled isonitrile-containing glutamic acid-urea derivative | |
| CN111138504B (en) | A kind of99mTc-CNPEDG complex and preparation method and application thereof | |
| CN112625065A (en) | Technetium-99 m marked FAPI (FAPI) derivative containing hydrazino nicotinamide and archaea, and preparation method and application thereof | |
| CN111518137B (en) | Technetium-99 m marked isonitrile-containing amino acid derivative and preparation method and application thereof | |
| Saccomanni et al. | Design, synthesis and preliminary evaluation of 18F-labelled 1, 8-naphthyridin-and quinolin-2-one-3-carboxamide derivatives for PET imaging of CB2 cannabinoid receptor | |
| US20080274046A1 (en) | Radiolabelling | |
| CN102126985B (en) | 18F labelled precursor compound and preparation method and application thereof | |
| Choi et al. | Synthesis and evaluation of an 125I-labeled azide prosthetic group for efficient and bioorthogonal radiolabeling of cyclooctyne-group containing molecules using copper-free click reaction | |
| CN103254139A (en) | Novel <18>F marked 4-aminoquinazoline derivatives, and preparation methods and tumor PET development application thereof | |
| Ruan et al. | Radiosynthesis and evaluation of novel [99mTc (I)]+ and [99mTc (I)(CO) 3]+ complexes with a 4‐nitroimidazole isocyanide for imaging tumor hypoxia | |
| CN105963724B (en) | A kind of radiolabeled tumor developer, preparation method and application | |
| CN101891791B (en) | Derivate for labeling bile acid and reference compound, preparation method and application thereof | |
| CN113583066B (en) | Mannose derivative and application thereof | |
| CN114031652B (en) | Glucose derivative containing cyclohexane and application thereof | |
| Xu et al. | Synthesis and biological evaluation of 7-(2-Chlorophenylamino)-5-((2-[18F] fluoro-ethyoxy) methyl) pyrazolo [1, 5-a] pyrimidine-3-carbonitrile as PET tumor imaging agent | |
| CA2811688A1 (en) | Choline analogs as radiotracer | |
| CN113150032B (en) | Technetium-99 m labeled isonitrile-containing folic acid derivative and preparation method and application thereof | |
| CA2811092A1 (en) | Novel precursors of radiolabelled choline analog compounds | |
| CN105001274B (en) | Complex, the Preparation method and use of a kind of Glucosamine derived ligand compound and preparation method, three carbonyl Tc 99ms mark | |
| CN106084004B (en) | 18F click-labeled transferrin receptor targeting polypeptide T7 as well as preparation method 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 |