CN106317112B - Tetrazine kind compound and preparation method thereof, application - Google Patents
Tetrazine kind compound and preparation method thereof, application Download PDFInfo
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Abstract
The invention discloses tetrazine kind compounds and preparation method thereof, application, tetrazine kind compound is tetrazine phosphate, the tetrazine phosphate of the preparation method preparation can be applied to prepare tetrazine analog derivative through the invention, and the tetrazine analog derivative containing fluorophor can be used as bio-orthogonal fluorescence probe.Tetrazine analog derivative is prepared using tetrazine phosphate of the present invention as intermediate, the tetrazine analog derivative type not only prepared is more, and remote red near infrared emission wavelength and long stoke shift bio-orthogonal fluorescence probe can be suitable for, prepared bio-orthogonal fluorescence probe have specificity it is stronger, background signal is lower, fluorescence-activation it is sensitive efficiently etc. advantages.
Description
Technical field
The present invention relates to tetrazine kind compound preparation technical fields, and in particular to tetrazine kind compound and preparation method thereof,
Using.
Background technique
Tetrazine kind compound and its derivative have a more physiological activity, such as anti-inflammatory, analgesia, antiviral, antibacterial and anti-
Cancer.Wherein, the tetrazine analog derivative containing fluorophor can be used as bioluminescence orthogonal probes, tetrazine analog derivative and corresponding
Bio-orthogonal chemistry have many advantages, such as good bio-compatibility, internal stability, be swift in response be widely used in from
Molecule and subcellular structure level seek the important research fields such as the chemical biology of cell and organization life process, pharmacy.More
It is important that the intrinsic physicochemical properties of tetrazine kind compound, allow to be designed to bioluminescence orthogonal probes, such spy
The fluorescence signal of needle is only activated by bio-orthogonal reaction, so that unreacted probe background signal in organism is greatly reduced,
Reach the resolution ratio and detection sensitivity for improving image.
But it is limited by the tetrazine analog derivative that current some tetrazine kind compound intermediates are prepared, mostly
Derivative containing aromatic group, gained bioluminescence orthogonal probes to fluorescence be quenched be by chemical bond energy transfer (or
Foster Resonance energy transfer) Lai Shixian, i.e., after tetrazine kind compound and known fluorescent molecule are covalently attached, in excitation state
Energy is transferred to tetrazine receptor from fluorescent chromophore the Worker's Stadium donor and discharges in non-radiative form.Since mechanism and existing is quenched
The limitation of the factors such as the intrinsic physicochemical characteristic of tetrazine kind compound is only applicable to the fluorescence point in some visible light area at present
Son, the existing remote red near IR fluorescence molecule with longer absorbing wavelength are difficult to carry out fluorescent quenching, limit by above-mentioned mechanism
Existing tetrazine kind compound has been made in the application in the fields such as Form imaging.
Summary of the invention
The purpose of the present invention is to provide a kind of tetrazine kind compounds, solve existing tetrazine kind compound synthesis tetrazine class
Limited, the prepared tetrazine class bio-orthogonal fluorescence probe of derivant structure is only applicable to the fluorescent molecule in some visible light area
Problem.
In addition, the present invention also provides a kind of preparation methods of tetrazine kind compound, application.
The present invention is achieved through the following technical solutions:
Tetrazine kind compound, tetrazine kind compound are tetrazine phosphate, structural formula are as follows:
Its wherein, R1For-H, replace alkyl, unsubstituted alkyl, thienyl ,-
(CH2)xNHBoc、-(CH2)xNHt-Bu、-(CH2)xNHFmoc、-OH、-Cl、-Br、-I、-NH2, secondary amino group, tertiary amino, carboxyl,
Phenyl or phenethyl, wherein x=1-5.
The substitution alkyl refers to that the H in alkyl is replaced by other elements or group, unsubstituted alkyl refers to that H is not taken
Generation.
With R1For methyl, the structure of tetrazine phosphate is passed through1H NMR is characterized, wherein the chemistry of each hydrogen atom
It is displaced specific as follows:
1H NMR(500MHz,CHLOROFORM-D)δ4.24(s,4H,-OCH2CH3),3.99(s,2H,-CH2PO
(OEt)2),3.11(s,3H,-CH3),1.35(s,6H,-OCH2CH3).The structure of the target product is consistent with expected structure.
The preparation method of tetrazine kind compound, comprising the following steps:
1) it is, starting material with compound 1, obtains compound 2 through methyl ether hydrolysis；
2), compound 2 replaces to obtain compound 3 by bromine；
3), compound 3 reacts to obtain tetrazine phosphate with triethyl phosphate；
The structural formula of the compound 1 are as follows:The structural formula of compound 2 are as follows:The structural formula of compound 3 are as follows:
Work as R1When for methyl, compound 1- compound 3 is respectively 3- (methoxymethyl) -6-methyl-1, and 2,4,5-
Tetrazine, 6-methyl-1,2,4,5-tetrazin-3-yl)-methanol, 3- (bromomethyl) -6-methyl-
1,2,4,5-tetrazine。
With R1For methyl, the structure of compound 1- compound 3 passes through1H NMR is characterized, wherein each hydrogen atom
Chemical shift is specific as follows:
Compound 1:1H NMR(500MHz,CHLOROFORM-D)δ5.02(s,2H,-CH2OCH3),3.57(s,3H,-
CH2OCH3),3.06(s,3H,-CH3).The structure of the target product is consistent with expected structure；
Compound 2:1H NMR(500MHz,CHLOROFORM-D)δ5.25(s,2H,-CH2OH),3.65(s,1H,-
CH2OH),3.09(s,3H,-CH3).The structure of the target product is consistent with expected structure；
Compound 3:1H NMR(500MHz,CHLOROFORM-D)δ4.94(s,2H,-CH2Br),3.11(s,3H,-CH3)。
The structure of the target product is consistent with expected structure.
Further, the reducing agent that step 1) uses is the dichloromethane solution of Boron tribromide.
Specifically, in reaction flask, compound 1 is dissolved in dry methylene chloride (DCM), by the two of Boron tribromide
Chloromethanes solution is slowly added dropwise in reaction flask, and reaction solution is in N2After reacting 3h at -78-25 DEG C under protection, reaction solution is poured into water
In, liquid separation is shaken, washing removes solvent by the method for revolving and obtains compound 2.
Specific synthesis technology is as follows:
Further, bromine replaces the substance used to be N- bromo-succinimide in step 2), by compound 2, N- bromo
Succimide sequentially adds in solvent DCM, and triphenylphosphine reaction is added by several times.
Specifically, compound 2, N- bromo-succinimide (NBS) are dissolved in dry methylene chloride (DCM), it will
Triphenylphosphine (PPh3) be added in reaction flask in three times, it in 0-25 DEG C of reaction 2h, is washed with secondary water, DCM extraction, with anhydrous sulphur
Sour magnesium is dry, and excess of solvent is removed in vacuum, after being purified by silica gel column chromatography, obtains compound 3.
Specific synthesis technology is as follows:
Then by compound 3, triethyl phosphate, be dissolved in dichloroethanes (DEC), in N2Protect lower 100 DEG C of heating anti-
After answering 2h, extra solvent is removed in vacuum, carries out after purification, obtaining compound 4 through silica gel column chromatography.
Specific synthesis technology is as follows:
Further, compound 1 the preparation method comprises the following steps: with R1CN and MeOCH2CN is raw material, and dioxane, trifluoro is added
Methanesulfonic acid zinc and hydrazine reaction preparation.
Specifically, by R1CN and MeOCH2CN (methoxyacetonitrile) is dissolved in dry dioxane (Dioxane), is added
After entering trifluoromethanesulfonic acid zinc, hydrazine is slowly added dropwise, reaction solution is in N2Lower 65 DEG C of heating are protected to react.After 18 hours, reaction solution is fallen
Entering in sodium nitrite in aqueous solution, hydrochloric acid (10M) is slowly added at 0 DEG C to pH value of solution=2-3, water phase is extracted with ethyl acetate,
After organic phase anhydrous magnesium sulfate is dry, filtering and concentrating after being purified by silica gel column chromatography, obtains compound 1.
Specific synthesis technology is as follows:
The tetrazine phosphate is applied to prepare tetrazine analog derivative by the application of tetrazine kind compound.
Wherein, the tetrazine analog derivative containing fluorophor can be used as bio-orthogonal fluorescence probe.
Further, tetrazine analog derivative the preparation method comprises the following steps: the tetrazine phosphate is dissolved in RCHO a certain amount of
HWE reaction occurs under the action of alkali and catalyst for solvent, wherein R is alkane, alkene, alkynes, the C=1-10 of C=1-10
Alkane substituent, alkene substituent, alkynes substituent,
Wherein, R4For-H, alkyl, aryl or naphthenic base；R2、R3、R5And R6For-H, primary amino group, secondary amino group, tertiary amino, life
Object molecule, dyestuff, fluorophor, halogeno-group ,-CF3、-CCl3、-CBr3、-Cl3、-OH、-NH2、-OCHF2、-OCF3、-OCCl3、-
OCBr3、-OCl3、-OCHCl2、-OCHBr2、-OCHI2、-COOH、-CONH2、-NO2、-SH、-SO3H、-SO4H、-SO2NH2、-
NHNH2、-ONH2、-NHC(O)NHNH2、-NHC(O)NH2、-NHSO2H ,-NHOH ,-NHC (O) H or-NHC (O) OH；
Wherein on the N of primary amino group, secondary amino group and tertiary amino substituent group be alkyl, it is hetero atom alkyl, aryl, naphthenic base, miscellaneous
Cyclophane base, heterocycloall yl groups, replace alkyl, substituted aryl, substituted cycloalkyl, substituted heterocycle aryl, substituted heterocycle naphthenic base,
Boc protecting group or Fmoc protecting group；
The structural formula of the tetrazine analog derivative are as follows:
Specifically, Horner-is carried out by the different types of aldehyde compound of tetrazine phosphate (compound 4)
The tetrazine analog derivative with novel structure is prepared in Wadsworth-Emmons (HWE) reaction, and it includes remote for especially having
The tetrazine class bio-orthogonal fluorescence probe of red near infrared emission wavelength and long stoke shift.
Further, the molar ratio of tetrazine phosphate, RCHO and alkali is 1:0.8:1.1.
Applicant is proved by a large number of experiments: preparing tetrazine analog derivative yield with higher using above-mentioned molar ratio.
Further, alkali DBU/LiBr, tBuOK, Cs2CO3、NaH、KOH、Ba(OH)2、LiOCH(CF3)2Or n-BuLi.
Further, the solvent is THF, CH3CN, DMSO or DMF.
The compound of existing tetrazine class is used to synthesize the limited configurations of tetrazine analog derivative, mostly containing aromatic group
Derivative, i.e., the R group in the structural formula of above-mentioned tetrazine analog derivative are aromatic group.Also, existing tetrazine class biology is just
Fluorescence probe is handed over to be only applicable to the fluorescent molecule in some visible light area.
Tetrazine phosphate of the present invention is raw material tetrazine kind compound made of the reaction of 3 steps with compound 1, this four
Piperazine phosphate can occur HWE with various types of aldehyde compounds and react, and the tetrazine class with novel structure is prepared and spreads out
Biology, i.e. R group in the structural formula of tetrazine analog derivative not only can be aromatic group, can also be other long conjugated bodies
System can get by adjusting the type of R group with four including remote red near infrared emission wavelength and long stoke shift
Piperazine class bio-orthogonal fluorescence probe.
A series of fluorescence probes prepared by the present invention are that fluorescence is activated by bio-orthogonal chemical reaction, the activation of fluorescence
Be as caused by reacting the variation of molecular structure itself, and bio-orthogonal chemical reaction efficiently not by other substances in organism
Interference, thus with other kinds of fluorescence probe ratio, have specificity it is stronger, background signal is lower, fluorescence-activation it is sensitive efficiently
Etc. advantages.
Compared with prior art, the present invention having the following advantages and benefits:
1, tetrazine analog derivative is prepared using tetrazine phosphate of the present invention as intermediate, can obtained more with novelty
The tetrazine analog derivative of structure compensates for the tetrazine analog derivative limited configurations prepared by existing tetrazine kind compound, mostly
The defect of derivative containing aromatic group.
2, the tetrazine analog derivative that through the invention prepared by the method, the fluorescent emission group of bio-orthogonal fluorescence probe
The structure and substitution base type of donor are controllable, and can prepare has including remote red near infrared emission wavelength and long stoke
Be displaced biological tetrazine class bio-orthogonal fluorescence probe, compensate for existing tetrazine class bio-orthogonal fluorescence probe be only applicable to part can
The defect of the fluorescent molecule in light-exposed area.
3, the bio-orthogonal fluorescence probe and other kinds of fluorescence probe ratio prepared by the present invention has specificity more
By force, background signal is lower, the sensitive advantages such as efficiently of fluorescence-activation.
4, based on the orthogonal fluorescence probe of new bio prepared by the present invention, prepare corresponding nucleic acid, polypeptide probe can
For the early stage fluorescence detection to specific biological beacon relevant to major disease.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment, the present invention is made
Further to be described in detail, exemplary embodiment of the invention and its explanation for explaining only the invention, are not intended as to this
The restriction of invention.
Synthesize tetrazine phosphate and using tetrazine phosphate be intermediate synthesis tetrazine analog derivative technique it is as follows:
R1There are many kinds of the substituent groups selected with R, can not illustrate, only illustrate by taking following embodiment as an example of the invention one by one
Specific implementation process.
Embodiment 1:
In the present embodiment, R1For methyl, R is methyl.
The preparation method of tetrazine kind compound, comprising the following steps:
1), in 50mL round-bottomed flask, compound 1 (280mg, 2mmol) is dissolved in dry methylene chloride (1mL)
In, the dichloromethane solution (0.4M, 2mL) of Boron tribromide is slowly added dropwise in reaction flask, reaction solution is in N2- 78-25 under protection
After reacting 3h at DEG C, reaction solution is poured into water, shakes liquid separation, washing removes solvent by the method for revolving, obtains compound 2
(219mg), yield: 87%；
The structural formula of compound 1 are as follows:The structural formula of compound 2 are as follows:
Wherein, the preparation process of compound 1 are as follows:
In 50mL round-bottomed flask, by CH3CN (acetonitrile) (410mg, 10mmol) and methoxyacetonitrile (213mg, 3mmol)
It is dissolved in dry dioxane (1mL), hydrazine 3mL is slowly added dropwise after trifluoromethanesulfonic acid zinc (108mg, 0.3mmol) is added, instead
Answer liquid in N2Lower 65 DEG C of heating are protected to react.After 18 hours, reaction solution is poured into sodium nitrite in aqueous solution (5M, 20mL), in 0
Hydrochloric acid (10M) is slowly added at DEG C to pH value of solution=2, water phase is extracted with ethyl acetate (50mL × 3), organic phase anhydrous magnesium sulfate
After drying, filtering and concentrating after being purified by silica gel column chromatography, obtains compound 1 (303mg), yield: 71%；
2), in 50mL round-bottomed flask, by compound 2 (252mg, 2mmol), N- bromo-succinimide (391.6mg,
It 2.2mmol) is dissolved in dry methylene chloride (2.2ml), by triphenylphosphine (PPh3) (131mg, 0.5mmol) in three times plus
Enter in reaction flask, in 0-25 DEG C of reaction 2h, washed with secondary water, DCM extraction, dry with anhydrous magnesium sulfate, it is extra to be removed in vacuum
Solvent after being purified by silica gel column chromatography, obtains compound 3 (313mg), yield: 83%；
The structural formula of compound 3 are as follows:
3), in 50mL round-bottomed flask, by compound 3 (378mg, 2mmol), triethyl phosphate (418mg, 2.2mmol)
It is dissolved in dichloroethanes (DCE, 2mL), in N2After protecting lower 100 DEG C of heating reaction 2h, extra solvent is removed in vacuum, through silicon
Plastic column chromatography carries out after purification, obtaining compound 4 (6- methyl-1,2,4,5- tetrazine phosphates) 418mg, yield: 85%.
Compound 4 prepared by the present embodiment is 6- methyl-1,2,4,5- tetrazine phosphates, structural formula are as follows:
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative:
In N2Under protection, the compound 4 of 0.2mmol is added in the DMSO solution of 0.16mmol acetaldehyde, is added
LiOCH (the CF of 0.22mmol3)2, and 1.5h is reacted at 45 DEG C, extra solvent is removed in vacuum after reaction, then through thin layer
Chromatography (petroleum ether: ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield:
90%, the structural formula of tetrazine analog derivative 1 is as follows:
Embodiment 2:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: have following difference: alkali used is tBuOK.
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, 0.22mmol is added
TBuOK, and react 2h at 25 DEG C, be removed in vacuum extra solvent after reaction, then through thin-layered chromatography (petroleum ether:
Ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield: 35%.
Embodiment 3:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: have following difference: alkali used is Cs2CO3。
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, 0.22mmol is added
Cs2CO3, and react 2h at 25 DEG C, be removed in vacuum extra solvent after reaction, then through thin-layered chromatography (petroleum ether:
Ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield: 25%.
Embodiment 4:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: have following difference: alkali used is NaH.
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, 0.22mmol is added
NaH, and react 2h at 25 DEG C, extra solvent be removed in vacuum after reaction, then through thin-layered chromatography (petroleum ether: second
Acetoacetic ester=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield: 10%.
Embodiment 5:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: have following difference: alkali used is Ba (OH)2。
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, 0.22mmol is added
Ba (OH)2, and 2h is reacted at 25 DEG C, extra solvent is removed in vacuum after reaction, then through thin-layered chromatography (petroleum
Ether: ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield: 55%.
Embodiment 6
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: have following difference: alkali used is LiOCH (CF3)2。
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, 0.22mmol is added
LiOCH (CF3)2, and 2h is reacted at 25 DEG C, extra solvent is removed in vacuum after reaction, then through thin-layered chromatography (stone
Oily ether: ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield: 67%.
Embodiment 7
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: there is following difference: solvent for use DMF, alkali is LiOCH (CF3)2。
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the DMF solution of 0.16mmol acetaldehyde, 0.22mmol is added
LiOCH (CF3)2, and 2h is reacted at 25 DEG C, extra solvent is removed in vacuum after reaction, then through thin-layered chromatography (stone
Oily ether: ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield: 71%.
Embodiment 8
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: there is following difference: solvent for use CH3CN, alkali are LiOCH (CF3)2, reaction temperature is
10 DEG C, reaction time 3h.
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added to the CH of 0.16mmol acetaldehyde3In CN solution, it is added
LiOCH (the CF of 0.22mmol3)2, and 2h is reacted at 10 DEG C, extra solvent is removed in vacuum after reaction, then through thin layer color
Spectrometry (petroleum ether: ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative 1, yield:
53%.
Embodiment 9
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent.
Compared with Example 1: there is following difference: solvent for use THF, alkali DBU/LiBr, reaction temperature is 45 DEG C,
Reaction time 1.5h.
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative 1:
In N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, is sequentially added
The LiBr of the DBU of 0.22mmol, 0.22mmol, and 2h is reacted at 25 DEG C, extra solvent is removed in vacuum after reaction, then
Through thin-layered chromatography, (petroleum ether: ethyl acetate=2:1 isolates and purifies product, is then dried to obtain tetrazine analog derivative
1, yield: 65%.
Embodiment 10:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent, only R1Substitution corresponding with R
Base is different.
Compared with Example 1: there is following difference: R1For phenyl, R is benzyl.
In the present embodiment, using acetonitrile and methoxyacetonitrile as raw material, compound is prepared using above-mentioned synthesis technology
4, compound 4 is methyl-tetrazine phosphate, structural formula are as follows:
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative:
In N2Under protection, the compound 4 of 0.2mmol is added in the DMSO solution of the phenylacetaldehyde of 0.16mmol, is added
LiOCH (the CF of 0.22mmol3)2, and 1.5h is reacted at 45 DEG C, extra solvent is removed in vacuum after reaction, then through thin layer
Chromatography isolates and purifies product, is then dried to obtain tetrazine analog derivative 2, yield: 67%, tetrazine analog derivative 2
Structural formula is as follows:
Embodiment 11:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent, only R1Substitution corresponding with R
Base is different.
Compared with Example 1: there is following difference: R1For tert-butyl (tBu), R is isobutyl group.
In the present embodiment, it is prepared for raw material using above-mentioned synthesis technology with 3,3- nitrile dimethyl and methoxyacetonitrile
Compound 4 is obtained, compound 4 is methyl-tetrazine phosphate, structural formula are as follows:
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative:
In N2Under protection, the compound 4 of 0.2mmol is added in the DMSO solution of the isopentyl aldehyde of 0.16mmol, is added
LiOCH (the CF of 0.22mmol3)2, and 1.5h is reacted at 45 DEG C, extra solvent is removed in vacuum after reaction, then through column color
Spectrum isolates and purifies product, is then dried to obtain tetrazine analog derivative 3, yield: 62%, structural formula is as follows:
Embodiment 12:
The present embodiment is based on embodiment 1, and the preparation method of compound 1- compound 4 is consistent, only R1Substitution corresponding with R
Base is different.
Compared with Example 1: there is following difference: R1For methyl, the aldehyde material used for
In the present embodiment, using acetonitrile and methoxyacetonitrile as raw material, compound is prepared using above-mentioned synthesis technology
4, compound 4 is methyl-tetrazine phosphate, structural formula are as follows:
Compound 4 manufactured in the present embodiment is used to prepare tetrazine analog derivative:
In N2Under protection, 0.16mmol is added in the compound 4 of 0.2mmol's
In DMSO solution, the LiOCH (CF of 0.22mmol is added3)2, and 1.5h is reacted at 45 DEG C, it is extra to be removed in vacuum after reaction
Solvent, then product is isolated and purified through column chromatography, is then dried to obtain tetrazine analog derivative 4, yield: 55%, knot
Structure formula is as follows:
Solvent I used in the preparation of 9 compound 5 of embodiment 1- embodiment, alkali II, reaction time, reaction temperature, gained produce
Object yield is as shown in table 1:
Table 1
| Embodiment | Solvent | Reaction time (h) | Temperature (DEG C) | Alkali | Yield (%) |
| 1 | DMSO | 1.5 | 45 | LiOCH(CF3)2 | 90 |
| 2 | THF | 2 | 25 | tBuOK | 35 |
| 3 | THF | 2 | 25 | Cs2CO3 | 25 |
| 4 | THF | 2 | 25 | NaH | 10 |
| 5 | THF | 2 | 25 | Ba(OH)2 | 55 |
| 6 | THF | 2 | 25 | LiOCH(CF3)2 | 67 |
| 7 | DMF | 2 | 25 | LiOCH(CF3)2 | 71 |
| 8 | CH3CN | 2 | 10 | LiOCH(CF3)2 | 53 |
| 9 | THF | 1.5 | 45 | DBU/LiBr | 65 |
Have as shown in Table 1, can be realized using the method for the invention and prepare tetrazine analog derivative, wherein alkali is to tetrazine
The influence of analog derivative yield is maximum, uses LiOCH (CF3)2, DBU/LiBr and Ba (OH)2Yield with higher, especially
LiOCH(CF3)2.Influence of the solvent to tetrazine analog derivative yield is taken second place.Temperature and reaction time within the above range can be real
The preparation of existing tetrazine analog derivative.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (6)
1. a kind of preparation method of tetrazine phosphate, which comprises the following steps:
1), compound 1 is dissolved in reaction flask in dry methylene chloride, the dichloromethane solution of Boron tribromide is slow
In dropwise reaction bottle, reaction solution is in N2After reacting 3h at -78-25 DEG C under protection, reaction solution is poured into water, shakes liquid separation, water
It washes, solvent is removed by the method for revolving and obtains compound 2；
2), compound 2, N- bromo-succinimide are dissolved in dry methylene chloride, triphenylphosphine is added in three times
It in reaction flask, in 0-25 DEG C of reaction 2h, is washed with secondary water, DCM extraction is dry with anhydrous magnesium sulfate, is removed in vacuum extra molten
Agent after being purified by silica gel column chromatography, obtains compound 3；
3), by compound 3, triethyl phosphate, be dissolved in dichloroethanes, in N2After protecting lower 100 DEG C of heating reaction 2h, vacuum
Extra solvent is removed, carries out after purification, obtaining tetrazine phosphate through silica gel column chromatography；
The structural formula of the compound 1 are as follows:The structural formula of compound 2 are as follows:The structural formula of compound 3 are as follows:The structure of the tetrazine phosphate
Formula are as follows:Wherein, R1For-H, unsubstituted alkyl, thienyl ,-(CH2)xNHBoc、-
(CH2)xNHt-Bu、-(CH2)xNHFmoc、-OH、-Cl、-Br、-I、-NH2, secondary amino group, tertiary amino, carboxyl, phenyl or phenethyl,
Wherein, x=1-5.
2. a kind of preparation method of tetrazine phosphate according to claim 1, which is characterized in that the system of the compound 1
Preparation Method are as follows: with R1CN and MeOCH2CN is raw material, and dioxane, trifluoromethanesulfonic acid zinc and hydrazine reaction preparation is added.
3. a kind of application of tetrazine phosphate, which is characterized in that the tetrazine phosphate is using preparation method described in claim 1
The tetrazine phosphate is applied to prepare tetrazine analog derivative by preparation；
The structural formula of the tetrazine analog derivative isWherein, R1For-H, unsubstituted alkane
Base, thienyl ,-(CH2)xNHBoc、-(CH2)xNHt-Bu、-(CH2)xNHFmoc、-OH、-Cl、-Br、-I、-NH2, secondary amino group,
Tertiary amino, carboxyl, phenyl or phenethyl, wherein x=1-5, R are the alkane of C=1-10, the alkene of C=1-10, C=1-10
Alkynes,
Wherein, R2、R3、R5And R6For-H, primary amino group, secondary amino group, tertiary amino；
The tetrazine analog derivative the preparation method comprises the following steps: the tetrazine phosphate and RCHO are dissolved in solvent, in the effect of alkali
Lower generation HWE reaction；
The structural formula of the tetrazine phosphate are as follows:
4. the application of tetrazine phosphate according to claim 3, which is characterized in that the tetrazine phosphate, RCHO and alkali
Molar ratio be 1:0.8:1.1.
5. the application of tetrazine phosphate according to claim 3, which is characterized in that the alkali be DBU/LiBr, tBuOK,
Cs2CO3、NaH、KOH、Ba(OH)2、LiOCH(CF3)2Or n-BuLi.
6. the application of tetrazine phosphate according to claim 3, which is characterized in that the solvent is THF, CH3CN、DMSO
Or DMF.
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