CN106317112A - Tetrazine compound, method for preparing same and application of tetrazine compound - Google Patents
Tetrazine compound, method for preparing same and application of tetrazine compound Download PDFInfo
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Abstract
The invention discloses a tetrazine compound, a method for preparing the same and application of the tetrazine compound. The tetrazine compound is tetrazine phosphate ester. The tetrazine phosphate ester prepared by the aid of the method can be applied to preparing tetrazine derivatives, and the tetrazine derivatives with fluorescent groups can be used as biological orthogonal fluorescent probes. The tetrazine compound, the method and the application have the advantages that the tetrazine phosphate ester is used as an intermediate for preparing the tetrazine derivatives, accordingly, different varieties of tetrazine derivatives can be prepared from the tetrazine phosphate ester and can be applied to the biological orthogonal fluorescent probes with far infrared/near infrared emission wavelengths and long Stoke's shift, the prepared biological orthogonal fluorescent probes are high in specificity, low in background signal and sensitive and efficient in fluorescent activation, and the like.
Description
Technical field
The present invention relates to tetrazine kind compound preparing technical field, be specifically related to tetrazine kind compound and preparation method thereof,
Application.
Background technology
Tetrazine kind compound and derivant thereof have more physiologically active, such as antiinflammatory, analgesia, antiviral, antibacterial and anti-
Cancer.Wherein, the tetrazine analog derivative containing fluorophor can be as bioluminescence orthogonal probes, and tetrazine analog derivative is with corresponding
Bio-orthogonal chemistry there is bio-compatibility stability good, internal, the advantage such as be swift in response be widely used in from
Molecule and subcellular structure aspect seek the important research field such as cell and the chemical biology of organization life process, pharmacy.More
It is important that the intrinsic physicochemical properties of tetrazine kind compound so that it is can be designed as bioluminescence orthogonal probes, such is visited
The fluorescence signal of pin is only activated by bio-orthogonal reaction, thus unreacted probe background signal in organism is greatly reduced,
Reach to improve resolution and the detection sensitivity of image.
But, the tetrazine analog derivative prepared by some current tetrazine kind compound intermediate is limited, mostly is
Derivant containing aromatic group, gained bioluminescence orthogonal probes to the cancellation of fluorescence be all by chemical bond energy shift (or
Foster Resonance energy transfer) realize, after i.e. tetrazine kind compound is covalently bound with known fluorescence molecule, it is in excited state
Energy is transferred to tetrazine receptor from fluorescent chromophore the Worker's Stadium donor and discharges with radiationless form.Due to cancellation mechanism and existing
The restriction of the factors such as the intrinsic physicochemical characteristic of tetrazine kind compound, the fluorescence being only applicable to some visible light district at present divides
Son, the existing remote red near Infrared fluorescence molecule with longer absorbing wavelength is difficult to carry out fluorescent quenching by above-mentioned mechanism, limit
Make the application in fields such as Form imagings of the existing tetrazine kind compound.
Summary of the invention
It is an object of the invention to provide a kind of tetrazine kind compound, solve existing tetrazine kind compound synthesis tetrazine class
Limited, the prepared tetrazine class bio-orthogonal fluorescent probe of derivant structure is only applicable to the fluorescence molecule in some visible light district
Problem.
Additionally, the present invention also provides for the preparation method of a kind of tetrazine kind compound, application.
The present invention is achieved through the following technical solutions:
Tetrazine kind compound, tetrazine kind compound is tetrazine phosphate ester, and its structural formula is:
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.
Described replacement alkyl refers to that the H in alkyl is replaced by other element or group, unsubstituted alkyl refers to that H is not taken
Generation.
With R1As a example by methyl, the structure of tetrazine phosphate ester is passed through1H NMR characterizes, the chemistry of the most each hydrogen atom
Displacement is 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 this target product is consistent with expected structure.
The preparation method of tetrazine kind compound, comprises the following steps:
1), with compound 1 as initiation material, compound 2 is obtained through methyl ether hydrolysis；
2), compound 2 obtains compound 3 through bromine replacement；
3), compound 3 and triethyl phosphate react and obtain tetrazine phosphate ester；
The structural formula of described compound 1 is:The structural formula of compound 2 is:The structural formula of compound 3 is:
Work as R1During for methyl, compound 1-compound 3 respectively 3-(methoxymethyl)-6-methyl-1,2,4,5-
Tetrazine, 6-methyl-1,2,4,5-tetrazin-3-yl) methanol, 3-(bromomethyl)-6-methyl-
1,2,4,5-tetrazine。
With R1As a example by methyl, the structure of compound 1-compound 3 is passed through1H NMR characterizes, the most 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 this 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 this 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 this target product is consistent with expected structure.
Further, step 1) dichloromethane solution that reducing agent is Boron tribromide that uses.
Specifically, in reaction bulb, compound 1 is dissolved in dry dichloromethane (DCM), by the two of Boron tribromide
Chloromethanes solution is slowly added dropwise in reaction bulb, and reactant liquor is in N2After reacting 3h at-78-25 DEG C under protection, pour reactant liquor into water
In, shaking separatory, washing, the method steamed by rotation is removed solvent and obtains compound 2.
Concrete synthesis technique is as follows:
Further, step 2) in bromine to replace the material used be N-bromo-succinimide, by compound 2, N-bromo
Succimide is sequentially added in solvent DCM, adds triphenylphosphine reaction by several times.
Specifically, compound 2, N-bromo-succinimide (NBS) are dissolved in dry dichloromethane (DCM), will
Triphenylphosphine (PPh3) add in three times in reaction bulb, reacting 2h at 0-25 DEG C, wash with secondary water, DCM extracts, and uses anhydrous sulfur
Acid magnesium is dried, and vacuum removes excess of solvent, through silica gel column chromatography after purification, obtains compound 3.
Concrete synthesis technique is as follows:
Then by compound 3, triethyl phosphate, it is dissolved in dichloroethanes (DEC), at N2Protect lower 100 DEG C of heating anti-
After answering 2h, vacuum removes unnecessary solvent, after silica gel column chromatography is purified, obtains compound 4.
Concrete synthesis technique is as follows:
Further, the preparation method of compound 1 is: with R1CN and MeOCH2CN is raw material, adds dioxane, trifluoro
Prepared by methanesulfonic acid zinc and hydrazine reaction.
Specifically, by R1CN and MeOCH2CN (methoxyacetonitrile) is dissolved in dry dioxane (Dioxane), adds
After entering trifluoromethanesulfonic acid zinc, being slowly added dropwise hydrazine, reactant liquor is in N2Protect lower 65 DEG C of reacting by heating.After 18 hours, reactant liquor is fallen
Enter in sodium nitrite in aqueous solution, at 0 DEG C, be slowly added to hydrochloric acid (10M) be extracted with ethyl acetate to pH value of solution=2-3, aqueous phase,
Organic facies anhydrous magnesium sulfate is dried, and filtering and concentrating through silica gel column chromatography after purification, obtains compound 1.
Concrete synthesis technique is as follows:
The application of tetrazine kind compound, is applied to prepare tetrazine analog derivative by described tetrazine phosphate ester.
Wherein, the tetrazine analog derivative containing fluorophor can be as bio-orthogonal fluorescent probe.
Further, the preparation method of tetrazine analog derivative is: be dissolved in a certain amount of by described tetrazine phosphate ester with RCHO
, there is HWE reaction in solvent under the effect of alkali and catalyst, wherein, R is the alkane of C=1-10, alkene, alkynes, C=1-10
Alkane substituent, alkene substituent, alkynes substituent,
Wherein, R4For-H, alkyl, aryl or cycloalkyl；R2、R3、R5And R6For-H, primary amino radical, secondary amino group, tertiary amino, life
Thing 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 radical, secondary amino group and tertiary amino, substituent group is alkyl, hetero atom alkyl, aryl, cycloalkyl, miscellaneous
Cyclophane base, heterocycloall yl groups, replacement alkyl, substituted aryl, substituted cycloalkyl, substituted heterocycle aryl, substituted heterocycle cycloalkyl,
Boc protection group or Fmoc protection group；
The structural formula of described tetrazine analog derivative is:
Specifically, Horner is carried out by tetrazine phosphate ester (compound 4) different types of aldehyde compound
Wadsworth Emmons (HWE) reaction prepares the tetrazine analog derivative with novel structure, particularly has and includes far
The tetrazine class bio-orthogonal fluorescent probe of red near infrared emission wavelength and long stoke shift.
Further, the mol ratio of tetrazine phosphate ester, RCHO and alkali is 1:0.8:1.1.
Applicant is proved by lot of experiments: uses above-mentioned mol ratio to prepare tetrazine analog derivative and has higher yield.
Further, alkali is DBU/LiBr, tBuOK, Cs2CO3、NaH、KOH、Ba(OH)2、LiOCH(CF3)2Or n-BuLi.
Further, described solvent is THF, CH3CN, DMSO or DMF.
The compound of existing tetrazine class, for synthesizing the limited configurations of tetrazine analog derivative, mostly is containing aromatic group
Derivant, the R group in the structural formula of the most above-mentioned tetrazine analog derivative is aromatic group.Further, existing tetrazine class biology is just
Fluorescent probe is handed over to be only applicable to the fluorescence molecule in some visible light district.
Tetrazine phosphate ester of the present invention reacts, with compound 1, the tetrazine kind compound made for raw material by 3 steps, and these are four years old
Piperazine phosphate ester can occur HWE react with various types of aldehyde compounds, prepares and has the tetrazine class of novel structure and spread out
R group in biology, the i.e. structural formula of tetrazine analog derivative is possible not only to be aromatic group, it is also possible to be other long conjugated body
System, by adjusting the type of R group, can obtain to have and include remote red near infrared emission wavelength and the four of long stoke shift
Piperazine class bio-orthogonal fluorescent probe.
A series of fluorescent probes prepared by the present invention are to activate fluorescence by bio-orthogonal chemical reaction, the activation of fluorescence
It is to be caused by the change of the molecular structure of reaction own, and bio-orthogonal chemical reaction is not efficiently by other materials in organism
Interference, therefore with other kinds of fluorescent probe ratio, has specificity higher, and background signal is lower, fluorescence-activation is sensitive efficiently
Etc. advantage.
The present invention compared with prior art, has such advantages as and beneficial effect:
1, tetrazine analog derivative is prepared with tetrazine phosphate ester of the present invention for intermediate, it is possible to obtain and more there is novelty
The tetrazine analog derivative of structure, be compensate for the tetrazine analog derivative limited configurations prepared by existing tetrazine kind compound, mostly is
The defect of the derivant containing aromatic group.
2, the tetrazine analog derivative prepared by the method for the invention, the fluorescent emission group of its bio-orthogonal fluorescent probe
The structure of donor and substituent group type controllable, can prepare to have and include remote red near infrared emission wavelength and long stoke
Displacement biology tetrazine class bio-orthogonal fluorescent probe, compensate for existing tetrazine class bio-orthogonal fluorescent probe be only applicable to part can
The defect of the fluorescence molecule in Jian Guang district.
3, the bio-orthogonal fluorescent probe prepared by the present invention and other kinds of fluorescent probe ratio, have specificity more
By force, background signal is lower, the sensitive advantage such as efficiently of fluorescence-activation.
4, based on the orthogonal fluorescent probe of new bio prepared by the present invention, corresponding nucleic acid is prepared, polypeptide probe can
For the early stage fluoroscopic examination to the specific biological beacon relevant to major disease.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is made
Further describing in detail, the exemplary embodiment of the present invention and explanation thereof are only used for explaining the present invention, are not intended as this
The restriction of invention.
Synthesis tetrazine phosphate ester and with tetrazine phosphate ester for intermediate synthesis tetrazine analog derivative technique as follows:
R1The substituent group selected with R has a variety of, it is impossible to illustrate one by one, only illustrates the present invention's as a example by following example
Specific implementation process.
Embodiment 1:
In the present embodiment, R1For methyl, R is methyl.
The preparation method of tetrazine kind compound, comprises the following steps:
1), in 50mL round-bottomed flask, compound 1 (280mg, 2mmol) is dissolved in dry dichloromethane (1mL)
In, the dichloromethane solution (0.4M, 2mL) of Boron tribromide is slowly added dropwise in reaction bulb, reactant liquor is in N2-78 25 under protection
After reacting 3h at DEG C, being poured into water by reactant liquor, shake separatory, washing, the method steamed by rotation is removed solvent, obtains compound 2
(219mg), yield: 87%；
The structural formula of compound 1 is:The structural formula of compound 2 is:
Wherein, the preparation process of compound 1 is:
In 50mL round-bottomed flask, by CH3CN (acetonitrile) (410mg, 10mmol) and methoxyacetonitrile (213mg, 3mmol)
It is dissolved in dry dioxane (1mL), after adding trifluoromethanesulfonic acid zinc (108mg, 0.3mmol), is slowly added dropwise hydrazine 3mL, instead
Answer liquid in N2Protect lower 65 DEG C of reacting by heating.After 18 hours, reactant liquor is poured into (5M, 20mL) in sodium nitrite in aqueous solution, in 0
Being slowly added to hydrochloric acid (10M) at DEG C to pH value of solution=2, aqueous phase is extracted with ethyl acetate (50mL × 3), organic facies anhydrous magnesium sulfate
After drying, filtering and concentrating, through silica gel column chromatography after purification, obtain compound 1 (303mg), yield: 71%；
2), in 50mL round-bottomed flask, by compound 2 (252mg, 2mmol), N-bromo-succinimide (391.6mg,
2.2mmol) it is dissolved in dry dichloromethane (2.2ml), by triphenylphosphine (PPh3) (131mg, 0.5mmol) add in three times
Entering in reaction bulb, react 2h at 0-25 DEG C, wash with secondary water, DCM extracts, and is dried with anhydrous magnesium sulfate, and it is unnecessary that vacuum removes
Solvent, through silica gel column chromatography after purification, obtains compound 3 (313mg), yield: 83%；
The structural formula of compound 3 is:
3), in 50mL round-bottomed flask, by compound 3 (378mg, 2mmol), triethyl phosphate (418mg, 2.2mmol)
It is dissolved in dichloroethanes (DCE, 2mL), at N2After protecting lower 100 DEG C of reacting by heating 2h, vacuum removes unnecessary solvent, through silicon
After plastic column chromatography is purified, obtain compound 4 (6-methyl isophthalic acid, 2,4,5-tetrazine phosphate esters) 418mg, yield: 85%.
Compound 4 prepared by the present embodiment is 6-methyl isophthalic acid, 2,4,5-tetrazine phosphate esters, and its structural formula is:
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative:
At N2Under protection, the compound 4 of 0.2mmol is added in the DMSO solution of 0.16mmol acetaldehyde, add
LiOCH (the CF of 0.22mmol3)2, and at 45 DEG C, react 1.5h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer
Chromatography (petroleum ether: product is carried out isolated and purified by ethyl acetate=2:1, 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: there is following difference: alkali used is tBuOK.
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, add 0.22mmol
TBuOK, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer chromatography (petroleum ether:
Product is carried out isolated and purified by ethyl acetate=2:1, 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: there is following difference: alkali used is Cs2CO3。
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, add 0.22mmol
Cs2CO3, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer chromatography (petroleum ether:
Product is carried out isolated and purified by ethyl acetate=2:1, 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: there is following difference: alkali used is NaH.
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, add 0.22mmol
NaH, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer chromatography (petroleum ether: second
Product is carried out isolated and purified by acetoacetic ester=2:1, 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: there is following difference: alkali used is Ba (OH)2。
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, add 0.22mmol
Ba (OH)2, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer chromatography (oil
Ether: product is carried out isolated and purified by ethyl acetate=2:1, 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: there is following difference: alkali used is LiOCH (CF3)2。
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, add 0.22mmol
LiOCH (CF3)2, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer chromatography (stone
Oil ether: product is carried out isolated and purified by ethyl acetate=2:1, 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: having following difference: solvent for use is DMF, alkali is LiOCH (CF3)2。
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the DMF solution of 0.16mmol acetaldehyde, add 0.22mmol
LiOCH (CF3)2, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer chromatography (stone
Oil ether: product is carried out isolated and purified by ethyl acetate=2:1, 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 is CH3CN, alkali is LiOCH (CF3)2, reaction temperature is
10 DEG C, response time 3h.
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added the CH of 0.16mmol acetaldehyde3In CN solution, add
LiOCH (the CF of 0.22mmol3)2, and at 10 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer color
Spectrometry (petroleum ether: product is carried out isolated and purified by ethyl acetate=2:1, 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: having following difference: solvent for use is THF, alkali is DBU/LiBr, and reaction temperature is 45 DEG C,
Response time 1.5h.
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative 1:
At N2Under protection, the compound 4 of 0.2mmol is added in the THF solution of 0.16mmol acetaldehyde, be sequentially added into
The LiBr of the DBU of 0.22mmol, 0.22mmol, and at 25 DEG C, react 2h, reaction terminates final vacuum and removes unnecessary solvent, then
Through thin layer chromatography, (petroleum ether: product is carried out isolated and purified by ethyl acetate=2:1, 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, simply R1The replacement corresponding with R
Base is different.
Compared with Example 1: there is following difference: R1For phenyl, R is benzyl.
In the present embodiment, with acetonitrile and methoxyacetonitrile as raw material, above-mentioned synthesis technique is used to prepare compound
4, compound 4 is methyl-tetrazine phosphate ester, and its structural formula is:
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative:
At N2Under protection, in the DMSO solution of the hyacinthin that the compound 4 of 0.2mmol is added 0.16mmol, add
LiOCH (the CF of 0.22mmol3)2, and at 45 DEG C, react 1.5h, reaction terminates final vacuum and removes unnecessary solvent, then through thin layer
Product is carried out isolated and purified by chromatography, 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, simply R1The replacement corresponding with R
Base is different.
Compared with Example 1: there is following difference: R1For the tert-butyl group (tBu), R is isobutyl group.
In the present embodiment, with 3,3-nitrile dimethyl and methoxyacetonitrile are raw material, use above-mentioned synthesis technique to prepare
Obtaining compound 4, compound 4 is methyl-tetrazine phosphate ester, and its structural formula is:
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative:
At N2Under protection, in the DMSO solution of the isovaleral that the compound 4 of 0.2mmol is added 0.16mmol, add
LiOCH (the CF of 0.22mmol3)2, and at 45 DEG C, react 1.5h, reaction terminates final vacuum and removes unnecessary solvent, then through post color
Product is carried out isolated and purified by spectrum, is then dried to obtain tetrazine analog derivative 3, yield: 62%, and its 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, simply R1The replacement corresponding with R
Base is different.
Compared with Example 1: there is following difference: R1For methyl, the aldehyde material of employing is
In the present embodiment, with acetonitrile and methoxyacetonitrile as raw material, above-mentioned synthesis technique is used to prepare compound
4, compound 4 is methyl-tetrazine phosphate ester, and its structural formula is:
The compound 4 prepared by the present embodiment is used for preparing tetrazine analog derivative:
At N2Under protection, the compound 4 of 0.2mmol is added 0.16mmol's
In DMSO solution, add the LiOCH (CF of 0.22mmol3)2, and at 45 DEG C, react 1.5h, it is unnecessary that reaction terminates final vacuum removing
Solvent, then through column chromatography, product is carried out isolated and purified, is then dried to obtain tetrazine analog derivative 4, yield: 55%, its knot
Structure formula is as follows:
Solvent I used by the preparation of embodiment 1-embodiment 9 compound 5, alkali II, response time, reaction temperature, gained produce
Thing yield is as shown in table 1:
Table 1
| Embodiment | Solvent | Response 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 |
Having as shown in Table 1, use the method for the invention to be capable of preparing tetrazine analog derivative, wherein, alkali is to tetrazine
The impact of analog derivative yield is maximum, uses LiOCH (CF3)2, DBU/LiBr and Ba (OH)2There is higher yield, especially
LiOCH(CF3)2.The impact of tetrazine analog derivative yield is taken second place by solvent.Temperature and response time the most all can be real
The preparation of existing tetrazine analog derivative.
Above-described detailed description of the invention, has been carried out the purpose of the present invention, technical scheme and beneficial effect further
Describe in detail, be it should be understood that the detailed description of the invention that the foregoing is only the present invention, be not intended to limit the present invention
Protection domain, all within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. done, all should comprise
Within protection scope of the present invention.
Claims (10)
1. tetrazine kind compound, it is characterised in that tetrazine kind compound is tetrazine phosphate ester, its structural formula is:
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.
2. the preparation method of a tetrazine kind compound as claimed in claim 1, it is characterised in that comprise the following steps:
1), with compound 1 as initiation material, compound 2 is obtained through methyl ether hydrolysis；
2), compound 2 obtains compound 3 through bromine replacement；
3), compound 3 and triethyl phosphate react and obtain tetrazine phosphate ester；
The structural formula of described compound 1 is:The structural formula of compound 2 is:
The structural formula of compound 3 is:
Its preparation method of tetrazine kind compound the most according to claim 2, it is characterised in that step 1) reduction that uses
Agent is the dichloromethane solution of Boron tribromide.
Its preparation method of tetrazine kind compound the most according to claim 2, it is characterised in that step 2) in bromine replace adopt
Material be N-bromo-succinimide, by compound 2, N-bromo-succinimide is sequentially added in solvent DCM, adds by several times
Enter triphenylphosphine reaction.
Its preparation method of tetrazine kind compound the most according to claim 2, it is characterised in that the system of described compound 1
Preparation Method is: with R1CN and MeOCH2CN is raw material, adds dioxane, trifluoromethanesulfonic acid zinc and hydrazine reaction and prepares.
6. the application of a tetrazine kind compound as claimed in claim 1, it is characterised in that described tetrazine phosphate ester is applied
In preparing tetrazine analog derivative.
The application of tetrazine kind compound the most according to claim 6, it is characterised in that the preparation of described tetrazine analog derivative
Method is: with RCHO, described tetrazine phosphate ester is dissolved in solvent, occurs HWE to react under the effect of alkali and catalyst, wherein,
R be the alkane of C=1-10, alkene, alkynes, the alkane substituent of C=1-10, alkene substituent, alkynes substituent,
Wherein, R4For-H, alkyl, aryl or cycloalkyl；R2、R3、R5And R6For-H, primary amino radical, secondary amino group, tertiary amino, biology point
Son, 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 radical, secondary amino group and tertiary amino, substituent group is alkyl, hetero atom alkyl, aryl, cycloalkyl, heterocycle virtue
Base, heterocycloall yl groups, replacement alkyl, substituted aryl, substituted cycloalkyl, substituted heterocycle aryl, substituted heterocycle cycloalkyl, Boc protect
Protect base or Fmoc protection group；
The structural formula of described tetrazine analog derivative is:
The application of tetrazine kind compound the most according to claim 7, it is characterised in that described tetrazine phosphate ester, RCHO and
The mol ratio of alkali is 1:0.8:1.1.
The application of tetrazine kind compound the most according to claim 7, it is characterised in that described alkali be DBU/LiBr,
tBuOK、Cs2CO3、NaH、KOH、Ba(OH)2、LiOCH(CF3)2Or n-BuLi.
The application of tetrazine kind compound the most according to claim 7, it is characterised in that described solvent is THF, CH3CN、
DMSO or DMF.
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| CN112812074A (en) * | 2020-12-31 | 2021-05-18 | 四川大学华西医院 | Tetrazine compound and preparation method and application thereof |
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| CN114106027A (en) * | 2021-11-10 | 2022-03-01 | 四川大学华西医院 | Fluoroboron fluorescent dye-tetrazine fluorescent probe and preparation method and application thereof |
| CN114106027B (en) * | 2021-11-10 | 2023-11-17 | 四川大学华西医院 | Fluoroboron fluorescent dye-tetrazine fluorescent probe and preparation method and application thereof |
| CN115974892A (en) * | 2022-12-27 | 2023-04-18 | 四川大学华西医院 | Triazole tetrazine compound and preparation method and application thereof |
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