3-cyano-5-aryl-1, 2, 4-triazazine compound as well as preparation method and application thereof

Abstract

The invention provides a 3-cyano-5-aryl-1, 2, 4-triazinetriazine compound, which is shown as a formula (I) or a formula (II) in the specification, and also provides a preparation method of the 3-cyano-5-aryl-1, 2, 4-triazinetriazine compound. Compared with the prior art, the 3-cyano-5-aryl-1, 2, 4-triazazine compound provided by the invention can be subjected to a rapid one-pot cycloaddition / oxidation reaction with a tension cyclooctenol compound under a room-temperature air condition to generate a corresponding pyridine compound, the secondary rate constant K2 is as high as 710M <-1 >. S <-1 >, and the compound can be applied to the preparation of a compound with high yield. A novel small molecule probe is provided for chemical and biological research of biological orthogonal link reaction. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a 3-cyano-5-aryl-1,2,4-triazine compound, a preparation method and application thereof. Background Art

[0002] The specific labeling of biomacromolecules in vivo with small molecule probes through bioorthogonal ligation chemistry is of great significance for understanding the molecular basis of life (EM Sletten, CR Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974-6998 and K. Liang, JW Chin, Chem. Rev. 2014, 114, 4764-4806). In recent years, J.A. Prescher of the United States, M.E. Webb of the United Kingdom, and M. Vrabel of the Czech Republic have used 1,2,4-triazine as a small molecule probe to develop new bioorthogonal reactions by inverse electron-demand Diels-Alder cycloaddition (IED-DA) with strained trans-cyclooctenol compounds (J.A. Prescher, et al., J. Am. Chem. Soc. 2015, 137, 8388-8391 & M.E. Webb, et al., Chem. Eur. J. 2015, 21, 14376–14381 & M. Vrabel, et al., Chem. Sci. 2017, 8, 3593–3598). However, these 1,2,4-triazine are either electron-rich or have diaromatic substituents at the 3 and 6 positions of the ring, resulting in slow cycloaddition reactions with strained cyclooctenols at room temperature (secondary rate constants K2 ranged from 0.075 to 9.9 M -1 ·S -1 ), which lacks practicality in bioorthogonal chemical connection experiments. Subsequently, Ma Junan's team in my country developed trifluoromethyl 1,2,4-triazine compounds, which greatly improved the cycloaddition reaction rate with strained cyclooctenol compounds at room temperature (the secondary rate constant K2 reached 99.2M -1 ·s -1 )(J.-A.Ma, et al., ACS Catal. 2019, 9, 4600–4608). However, in order to improve the reaction efficiency, there is still an urgent need to design and synthesize new 1,2,4-triazine compounds for bioorthogonal ligation chemistry. Summary of the invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a 3-cyano-5-aryl-1,2,4-triazine compound, a preparation method and application thereof, wherein the 3-cyano-5-aryl-1,2,4-triazine compound has a high cycloaddition reaction rate with a strained cyclooctenol compound at room temperature.

[0004] The present invention provides a 3-cyano-5-aryl-1,2,4-triazine compound, as shown in formula (I) or formula (II):

[0005]

[0006] Wherein, Ar is selected from substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-20 heterocyclic group;

[0007] The substituents in the substituted C6-C20 aryl group and the substituted C3-C20 heterocyclic group are each independently selected from one or more of C1-C10 alkyl group, C1-C10 alkoxy group, C1-C10 haloalkyl group, halogen, cyano group and nitro group.

[0008] Preferably, Ar is selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C3-10 heterocyclic group;

[0009] The substituents in the substituted C6-C10 aryl group and the substituted C3-C10 heterocyclic group are independently selected from one or more of C1-C5 alkyl group, C1-C5 alkoxy group, C1-C5 haloalkyl group, halogen, cyano group and nitro group.

[0010] Preferably, Ar is selected from substituted or unsubstituted phenyl, substituted or unsubstituted furyl;

[0011] The substituents in the substituted phenyl group and the substituted furyl group are independently selected from one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen, cyano and nitro.

[0012] Preferably, one of the structures shown in Ia~Il and II-a~II-l:

[0013]

[0014] The present invention also provides a method for preparing a 3-cyano-5-aryl-1,2,4-triazine compound, comprising the following steps:

[0015] The compound represented by formula (IV) is subjected to a dehydration reaction under alkaline conditions using trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (I);

[0016] Alternatively, the compound represented by formula (VI) is subjected to a dehydration reaction under alkaline conditions using trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (II);

[0017]

[0018] Wherein, Ar is selected from substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-20 heterocyclic group;

[0019] The substituents in the substituted C6-C20 aryl group and the substituted C3-C20 heterocyclic group are each independently selected from one or more of C1-C10 alkyl group, C1-C10 alkoxy group, C1-C10 haloalkyl group, halogen, cyano group and nitro group.

[0020] Preferably, the alkaline condition is provided by an organic base; the molar ratio of the trifluoroacetic anhydride to the organic base is 1:1 to 1:3.

[0021] Preferably, the compound represented by formula (IV) is prepared according to the following steps:

[0022] The compound represented by formula (III) is reacted with an alcohol solution of aqueous ammonia to obtain a compound represented by formula (IV);

[0023] And / or, the compound represented by formula (VI) is prepared according to the following steps:

[0024] The compound represented by formula (V) is reacted with an alcohol solution of aqueous ammonia to obtain a compound represented by formula (VI);

[0025]

[0026] Wherein, R and R' are each independently selected from C1-C5 alkyl groups.

[0027] Preferably, the mass concentration of the ammonia water is 25% to 38%; and the volume ratio of the ammonia water to the alcohol solvent in the alcohol solution of the ammonia water is 1:0.5 to 1:4.

[0028] The present invention also provides a use of the above 3-cyano-5-aryl-1,2,4-triazine compound as a small molecule probe.

[0029] The present invention provides a 3-cyano-5-aryl-1,2,4-triazine compound, as shown in formula (I) or formula (II). Compared with the prior art, the 3-cyano-5-aryl-1,2,4-triazine compound provided by the present invention can react with a strained cyclooctenol compound under room temperature and air conditions to undergo a rapid one-pot cycloaddition / oxidation reaction to generate a corresponding pyridine compound, and the secondary rate constant K2 is as high as 710M -1 ·s -1 , providing a new type of small molecule probe for chemical biology research on bioorthogonal linking reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound If obtained in Example 6 of the present invention;

[0031] Figure 2 is the nuclear magnetic resonance fluorine spectrum of the compound If obtained in Example 6 of the present invention;

[0032] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of compound Ij obtained in Example 10 of the present invention;

[0033] Figure 4 This is the nuclear magnetic resonance fluorine spectrum of compound Ij obtained in Example 10 of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] The present invention provides a 3-cyano-5-aryl-1,2,4-triazine compound, as shown in formula (I) or formula (II):

[0036]

[0037] Wherein, Ar is a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 heterocyclic group; the substituents in the substituted C6-C20 aryl group and the substituted C3-C20 heterocyclic group are each independently one or more of a C1-C10 alkyl group, a C1-C10 alkoxy group, a C1-C10 haloalkyl group, a halogen, a cyano group and a nitro group.

[0038] In a specific embodiment provided by the present invention, Ar is a substituted or unsubstituted C6-C15 aryl group, a substituted or unsubstituted C3-C15 heterocyclic group; the substituents in the substituted C6-C15 aryl group and the substituted C3-C15 heterocyclic group are each independently one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen, cyano and nitro, preferably one or more of C1-C5 alkyl, C1-C5 alkoxy, C1-C5 haloalkyl, halogen, cyano and nitro. The present invention further comprises one or more of a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, a halogen group, a cyano group and a nitro group, and more preferably one or more of a methyl group, an ethyl group, a methoxy group, an ethoxy group, a halomethyl group, a haloethyl group, a halogen group, a cyano group and a nitro group; the halogen group may be any halogen group known to those skilled in the art without any particular limitation, and in the present invention, it may be one or more of fluorine, chlorine and bromine; the heteroatom group may be any heteroatom group known to those skilled in the art without any particular limitation, and in the present invention, it may be nitrogen, oxygen or sulfur.

[0039] In a specific embodiment provided by the present invention, Ar is a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C3-C10 heterocyclic group; the substituents in the substituted C6-C10 aryl group and the substituted C3-C10 heterocyclic group are each independently one or more of C1-C10 alkyl group, C1-C10 alkoxy group, C1-C10 haloalkyl group, halogen, cyano group and nitro group, preferably one or more of C1-C5 alkyl group, C1-C5 alkoxy group, C1-C5 haloalkyl group, halogen, cyano group and nitro group. The present invention further comprises one or more of a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, a halogen group, a cyano group and a nitro group, and more preferably one or more of a methyl group, an ethyl group, a methoxy group, an ethoxy group, a halomethyl group, a haloethyl group, a halogen group, a cyano group and a nitro group; the halogen group may be any halogen group known to those skilled in the art without any particular limitation, and in the present invention, it may be one or more of fluorine, chlorine and bromine; the heteroatom group may be any heteroatom group known to those skilled in the art without any particular limitation, and in the present invention, it may be nitrogen, oxygen or sulfur.

[0040] In a specific embodiment provided by the present invention, Ar is a substituted or unsubstituted phenyl group, a substituted or unsubstituted furyl group; the substituents in the substituted phenyl group and the substituted furyl group are each independently one or more of a C1-C10 alkyl group, a C1-C10 alkoxy group, a C1-C10 haloalkyl group, a halogen, a cyano group and a nitro group, preferably one or more of a C1-C5 alkyl group, a C1-C5 alkoxy group, a C1-C5 haloalkyl group, a halogen, a cyano group and a nitro group, more preferably a C1-C3 alkyl group. The invention further comprises one or more of a C1-C3 alkoxy group, a C1-C3 haloalkyl group, a halogen, a cyano group and a nitro group, and more preferably one or more of a methyl group, an ethyl group, a methoxy group, an ethoxy group, a halomethyl group, a haloethyl group, a halogen, a cyano group and a nitro group; the halogen group may be any halogen group known to those skilled in the art without any particular limitation, and in the present invention, it is preferably one or more of fluorine, chlorine and bromine; the heteroatom in the heterocyclic group may be any heteroatom group known to those skilled in the art without any particular limitation, and in the present invention, it is preferably nitrogen, oxygen or sulfur.

[0041] In a specific embodiment provided by the present invention, Ar is phenyl, 4-methylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-nitrophenyl, 2,4-dinitrobenzene or 2-furanyl.

[0042] In a specific embodiment provided by the present invention, the 3-cyano-5-aryl-1,2,4-triazine compound is one of the structures shown in Ia~Il and II-a~II-l:

[0043]

[0044] The 3-cyano-5-aryl-1,2,4-triazine compound provided by the present invention can react with a strained cyclooctenol compound in a rapid one-pot cycloaddition / oxidation reaction under room temperature and air conditions to generate a corresponding pyridine compound, and the secondary rate constant K2 is as high as 710M -1 ·s -1 , providing a new type of small molecule probe for chemical biology research on bioorthogonal linking reactions.

[0045] The present invention also provides a method for preparing the above-mentioned 3-cyano-5-aryl-1,2,4-triazine compound, comprising the following steps: subjecting the compound represented by formula (IV) to a dehydration reaction under alkaline conditions using trifluoroacetic anhydride as a dehydrating agent to obtain the 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (I);

[0046] Alternatively, the compound represented by formula (VI) is subjected to a dehydration reaction under alkaline conditions using trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (II);

[0047]

[0048] Wherein, Ar is a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 heterocyclic group; the substituents in the substituted C6-C20 aryl group and the substituted C3-C20 heterocyclic group are each independently selected from one or more of C1-C10 alkyl group, C1-C10 alkoxy group, C1-C10 haloalkyl group, halogen, cyano group and nitro group.

[0049] The present invention has no special limitation on the sources of all raw materials, and any raw materials available on the market can be used; the Ar is the same as described above and will not be described in detail here.

[0050] In a specific embodiment provided by the present invention, the compound represented by formula (IV) is prepared according to the following steps: the compound represented by formula (III) is reacted with an alcohol solution of ammonia water to obtain a compound represented by formula (IV); the mass concentration of the ammonia water is preferably 25% to 38%, more preferably 25% to 35%; the volume ratio of ammonia water to alcohol solvent in the alcohol solution of ammonia water is preferably 1:0.5 to 1:4, more preferably 1:0.5 to 1:3, more preferably 1:0.5 to 1:2, more preferably 1:0.5 to 1:1.5, and most preferably 1:0.8 to 1:1; the alcohol solvent is an alcohol solvent well known to those skilled in the art, and there is no special limitation. In the present invention, methanol is preferably used. ; In the present invention, it is preferred to first mix the compound represented by formula (III) with an alcohol solvent, and then add ammonia water; the reaction is preferably carried out at room temperature; the reaction time is preferably 4 to 10 hours, more preferably 4 to 8 hours, and more preferably 6 hours; after the reaction, the alcohol solvent is preferably removed, and then dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a desiccant, and then the solvent is removed to obtain a compound represented by formula (IV); the methods for removing the alcohol solvent and removing the solvent are methods well known to those skilled in the art and are not particularly limited. In the present invention, thin-film rotary evaporation is preferred; the number of washings with saturated brine is preferably 2 to 3 times; the desiccant is preferably anhydrous sodium sulfate.

[0051]

[0052] Among them, R is a C1-C5 alkyl group, preferably a C1-C3 alkyl group, and more preferably a methyl group or an ethyl group.

[0053] The compound represented by formula (IV) is subjected to a dehydration reaction under alkaline conditions with trifluoroacetic anhydride (TFAA) as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (I); in the present invention, the compound represented by formula (IV) is preferably dried and then subjected to a dehydration reaction under alkaline conditions with trifluoroacetic anhydride (TFAA) as a dehydrating agent; the drying is preferably high vacuum drying; the high vacuum drying time is preferably 8 to 20 hours, more preferably 10 to 15 hours, and more preferably 12 hours; the alkaline conditions are preferably provided by an organic base; the organic base is an organic base well known to those skilled in the art, namely Yes, there is no special limitation, and triethylamine (TEA) is preferred in the present invention; the molar ratio of trifluoroacetic anhydride to the organic base is preferably 1:1 to 1:3, more preferably 1:1.5 to 1:3, more preferably 1:2 to 1:3, and most preferably 1:2.5 to 1:2.7; the dehydration reaction is preferably carried out in an organic solvent; the organic solvent is an organic solvent well known to those skilled in the art, and there is no special limitation, and tetrahydrofuran is preferred in the present invention; in the present invention, the compound represented by formula (IV) is preferably first mixed with an organic solvent and an organic base, and then trifluoroacetic anhydride is added under low temperature conditions, and the temperature is increased to carry out the dehydration reaction; The mixing and dehydration reaction is preferably carried out in a protective atmosphere; the protective atmosphere is any protective atmosphere well known to those skilled in the art and is not particularly limited. In the present invention, nitrogen is preferably used; the temperature of the low temperature condition is preferably 0°C to 3°C; the dehydration reaction is preferably carried out at room temperature; the dehydration reaction time is preferably 8 to 20 hours, more preferably 10 to 15 hours, and more preferably 12 hours; after the dehydration reaction is completed, water is preferably added to quench the reaction, and after removing the organic solvent, dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a desiccant, and the solvent is removed, and purified to obtain 3-cyanoacetate represented by formula (I) The method of removing the organic solvent and the solvent removal is a method well known to those skilled in the art and is not particularly limited. In the present invention, thin-film rotary evaporation is preferred. The number of washings with saturated brine is preferably 2 to 3 times. The desiccant is preferably anhydrous sodium sulfate. The purification is preferably silica gel column chromatography purification, and more preferably rapid silica gel column chromatography purification. The elution solvent used for the purification is preferably petroleum ether and ethyl acetate. In the present invention, petroleum ether and ethyl acetate in a volume ratio of 20:1 are preferably used as the eluent, and then petroleum ether and ethyl acetate in a volume ratio of 10:1 are used as the eluent.

[0054] In a specific embodiment provided by the present invention, the 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (I) is reacted according to the following reaction formula:

[0055]

[0056] In a specific embodiment provided by the present invention, the compound represented by formula (VI) is prepared according to the following steps: the compound represented by formula (V) is reacted with an alcohol solution of ammonia water to obtain a compound represented by formula (VI); the mass concentration of the ammonia water is preferably 25% to 38%, more preferably 25% to 35%; the volume ratio of ammonia water to alcohol solvent in the alcohol solution of ammonia water is preferably 1:0.5 to 1:4, more preferably 1:0.5 to 1:3, more preferably 1:0.5 to 1:2, more preferably 1:0.5 to 1:1.5, and most preferably 1:0.8 to 1:1; the alcohol solvent is a solvent known to those skilled in the art. Any well-known alcohol solvent can be used without special restrictions. Methanol is preferred in the present invention. In the present invention, it is preferred to first mix the compound represented by formula (V) with the alcohol solvent, and then add ammonia water dropwise. The reaction is preferably carried out at room temperature. The reaction time is preferably 4 to 10 hours, more preferably 4 to 8 hours, and more preferably 6 hours. After the reaction, the alcohol solvent is preferably removed, and then dichloromethane is added to obtain an organic phase. The organic phase is washed with saturated brine, dried with a desiccant, and then the solvent is removed to obtain a compound represented by formula (VI). The number of times the saturated brine is washed is preferably 2 to 3 times. The desiccant is preferably anhydrous sodium sulfate.

[0057]

[0058] Among them, R' is a C1-C5 alkyl group, preferably a C1-C3 alkyl group, and more preferably a methyl group or an ethyl group.

[0059] The compound represented by formula (VI) is subjected to a dehydration reaction under alkaline conditions with trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (II); in the present invention, the compound represented by formula (VI) is preferably dried and then subjected to a dehydration reaction under alkaline conditions with trifluoroacetic anhydride (TFAA) as a dehydrating agent; the drying is preferably high vacuum drying; the time for the high vacuum drying is preferably 8 to 20 hours, more preferably 10 to 15 hours, and more preferably 12 hours; the alkaline conditions are preferably provided by an organic base; the organic base is an organic base well known to those skilled in the art, and there is no particular limitation. The molar ratio of trifluoroacetic anhydride to the organic base is preferably 1:1 to 1:3, more preferably 1:1.5 to 1:3, more preferably 1:2 to 1:3, and most preferably 1:2.5 to 1:2.7; the dehydration reaction is preferably carried out in an organic solvent; the organic solvent is any organic solvent well known to those skilled in the art, and is not particularly limited, and is preferably tetrahydrofuran in the present invention; in the present invention, the compound represented by formula (VI) is preferably first mixed with an organic solvent and an organic base, and then trifluoroacetic anhydride is added under low temperature conditions, and the temperature is increased to carry out the dehydration reaction; the mixture The dehydration reaction is preferably carried out in a protective atmosphere; the protective atmosphere is any protective atmosphere well known to those skilled in the art and is not particularly limited. In the present invention, nitrogen is preferably used; the temperature of the low temperature condition is preferably 0°C to 3°C; the dehydration reaction is preferably carried out at room temperature; the dehydration reaction time is preferably 8 to 20 hours, more preferably 10 to 15 hours, and more preferably 12 hours; after the dehydration reaction is completed, water is preferably added to quench the reaction, and after removing the organic solvent, dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a desiccant, and the solvent is removed, and purified to obtain 3-cyanoacetate represented by formula (II) The method of removing the organic solvent and the solvent removal is a method well known to those skilled in the art and is not particularly limited. In the present invention, thin-film rotary evaporation is preferred. The number of washings with saturated brine is preferably 2 to 3 times. The desiccant is preferably anhydrous sodium sulfate. The purification is preferably silica gel column chromatography purification, and more preferably rapid silica gel column chromatography purification. The elution solvent used for the purification is preferably petroleum ether and ethyl acetate. In the present invention, petroleum ether and ethyl acetate in a volume ratio of 15:1 are preferably used as the eluent, and then petroleum ether and ethyl acetate in a volume ratio of 5:1 are used as the eluent.

[0060] In a specific embodiment provided by the present invention, the 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (II) is reacted according to the following reaction formula:

[0061]

[0062] The present invention also provides a use of the above 3-cyano-5-aryl-1,2,4-triazine compound as a small molecule probe.

[0063] In a specific embodiment provided by the present invention, the 3-cyano-5-aryl-1,2,4-triazine compound can act as a diene donor and undergo a rapid cycloaddition reaction with a strained cyclooctenol compound to generate a pyridine compound, so the 3-cyano-5-aryl-1,2,4-triazine can be used as a small molecule probe for bioorthogonal linking reactions.

[0064] Furthermore, the above cycloaddition reaction is carried out at room temperature and in air.

[0065] In order to further illustrate the present invention, a 3-cyano-5-aryl-1,2,4-triazine compound provided by the present invention, its preparation method and application are described in detail below in conjunction with examples.

[0066] The reagents used in the following examples are all commercially available. Flash column chromatography used Qingdao Ocean Chemical 200-300 mesh standard silica gel; thin layer chromatography used Qingdao Ocean Chemical 0.20 mm standard plate; nuclear magnetic resonance spectroscopy data (NMR) were obtained by testing with a Bruker 400 MHz nuclear magnetic resonance instrument, with tetramethylsilane as the internal standard and deuterated chloroform as the solvent (s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet).

[0067] Example 1: Preparation of 3-cyano-5-phenyl-6-(trifluoromethyl)-1,2,4-triazine Ia

[0068]

[0069] Take a 25mL round-bottom flask, add 6-(trifluoromethyl)-5-phenyl-3-carboxylic acid methyl ester (1.42g, 5.0mmol) and 5mL methanol at room temperature, then add 25% concentrated ammonia (5ml) dropwise, react at room temperature for 6h, and the reaction is complete when detected by thin layer chromatography; remove methanol by thin film rotary evaporation, add 30ml of dichloromethane to the mixture, transfer to a separatory funnel for separation, wash the organic phase twice with saturated brine (10mL×2), dry with anhydrous sodium sulfate, and remove the solvent by thin film rotary evaporation to obtain the intermediate amide. Transfer it to a 25mL Schlenk bottle, dry it under high vacuum for 12 hours, replace it with nitrogen, then add anhydrous THF (8mL) and triethylamine (2.02g, 20.0mmol), place it at 0℃, add trifluoroacetic anhydride (1.58g, 7.5mmol) dropwise, and then return to room temperature for reaction for 12h. After the reaction was completed, 5 mL of water was added to quench the reaction; tetrahydrofuran was removed by thin film rotary evaporation, 30 mL of dichloromethane was added to the mixture, and the mixture was transferred to a separatory funnel for separation. The organic phase was washed twice with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed by thin film rotary evaporation. The crude product was separated by rapid silica gel column chromatography (first using 20:1 volume ratio of petroleum ether and ethyl acetate as elution solvent, and then using 10:1 volume ratio of petroleum ether and ethyl acetate as elution solvent) to obtain product Ia (0.94 g, 75% yield). The compound was analyzed by nuclear magnetic resonance, and the obtained 1 HNMR(600MHz, CDCl3)δ7.49-7.53(m,3H),7.72(d,2H); 19 F NMR(565MHz, CDCl3)δ--61.30(s,3F).

[0070] Example 2: Preparation of 3-cyano-5-(4-methylphenyl)-6-(trifluoromethyl)-1,2,4-triazine Ib

[0071]

[0072] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(4-methylphenyl)-3-carboxylic acid methyl ester (1.49 g, 5.0 mmol) was used as a raw material to obtain product Ib (1.03 g, 78% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ2.33(s,3H),7.37(d,2H),7.76(d,2H); 19 F NMR (565MHz, CDCl3) δ–61.19 (s, 3F).

[0073] Example 3: Preparation of 3-cyano-5-(4-methoxyphenyl)-6-(trifluoromethyl)-1,2,4-triazine Ic

[0074]

[0075] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(4-methoxyphenyl)-3-carboxylic acid methyl ester (1.57 g, 5.0 mmol) was used as a raw material to obtain product Ic (1.13 g, 81% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ3.83(s,3H),7.04(d,2H),7.85(d,2H); 19 F NMR(565MHz, CDCl3)δ–61.22(s,3F).

[0076] Example 4: Preparation of 3-cyano-5-(4-fluorophenyl)-6-(trifluoromethyl)-1,2,4-triazine Id

[0077]

[0078] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(4-fluorophenyl)-3-carboxylic acid methyl ester (1.51 g, 5.0 mmol) was used as a raw material to obtain product Id (0.83 g, 62% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 HNMR(600MHz, CDCl3)δ7.32(t,2H),7.81-7.85(m,2H); 19 F NMR (565MHz, CDCl3) δ – 61.30 (s, 3F), – 105.81-105.88 (m, F).

[0079] Example 5: Preparation of 3-cyano-5-(4-chlorophenyl)-6-(trifluoromethyl)-1,2,4-triazine Ie

[0080]

[0081] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(4-chlorophenyl)-3-carboxylic acid methyl ester (1.59 g, 5.0 mmol) was used as a raw material to obtain product Ie (0.80 g, 56% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.73(d,2H),7.51(d,2H); 19F NMR (565MHz, CDCl3) δ–61.00 (s, 3F).

[0082] Example 6: Preparation of 3-cyano-5-(4-bromophenyl)-6-(trifluoromethyl)-1,2,4-triazine If

[0083]

[0084] A method similar to that of Example 1 was used, with methyl 6-(trifluoromethyl)-5-(4-bromophenyl)-3-carboxylate (1.81 g, 5.0 mmol) as starting material to obtain product If (0.95 g, 58% yield).

[0085] The compound was analyzed by nuclear magnetic resonance, and the nuclear magnetic resonance hydrogen spectrum was obtained as follows: Figure 1 As shown; get the result 1 H NMR (600MHz,CDCl3)δ7.77(d,2H),7.71(d,2H); the NMR fluorine spectrum was obtained as follows Figure 2 As shown; get the result 19 F NMR (565MHz, CDCl3) δ–61.47 (s, 3F).

[0086] Example 7: Preparation of 3-cyano-5-(4-iodophenyl)-6-(trifluoromethyl)-1,2,4-triazine Ig

[0087]

[0088] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(4-iodophenyl)-3-carboxylic acid methyl ester (2.05 g, 5.0 mmol) was used as a raw material to obtain product Ig (0.98 g, 52% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.60-7.73 (m, 4H); 19 FNMR (565MHz, CDCl3) δ – 59.92 (s, 3F).

[0089] Example 8: Preparation of 3-cyano-5-[(4-trifluoromethyl)phenyl]-6-(trifluoromethyl)-1,2,4-triazine Ih

[0090]

[0091] Using a method similar to Example 1, 6-(trifluoromethyl)-5-[(4-trifluoromethyl)phenyl]-3-carboxylic acid methyl ester (1.76 g, 5.0 mmol) was used as a raw material to obtain product Ih (0.65 g, 41% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.83(d,2H),7.95(d,2H); 19 F NMR (565MHz, CDCl3) δ – 61.21 (s, 3F), – 63.23 (s, 3F).

[0092] Example 9: Preparation of 3-cyano-5-(4-cyanophenyl)-6-(trifluoromethyl)-1,2,4-triazine Ii

[0093]

[0094] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(4-cyanophenyl)-3-carboxylic acid methyl ester (1.54 g, 5.0 mmol) was used as a raw material to obtain product Ii (0.36 g, 26% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.89 (dd, 4H); 19 FNMR(565MHz, CDCl3)δ–61.17(s,3F).

[0095] Example 10: Preparation of 3-cyano-5-(4-nitrophenyl)-6-(trifluoromethyl)-1,2,4-triazine

[0096]

[0097] A method similar to that of Example 1 was used, with 6-(trifluoromethyl)-5-(4-nitrophenyl)-3-carboxylic acid methyl ester (1.64 g, 5.0 mmol) as starting material to obtain product Ij (0.27 g, 18% yield).

[0098] The compound was analyzed by nuclear magnetic resonance, and its nuclear magnetic resonance hydrogen spectrum was obtained as follows: Figure 3 As shown; get the result 1 H NMR (600MHz,CDCl3)δ8.49(d,2H),8.01(d,2H); Its NMR fluorine spectrum is as follows Figure 4 As shown; get the result 19 F NMR(565MHz, CDCl3)δ–61.21(s,3F).

[0099] Example 11: Preparation of 3-cyano-5-(2,4-dinitrophenyl)-6-(trifluoromethyl)-1,2,4-triazine Ik

[0100]

[0101] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(2,4-dinitrophenyl)-3-methyl carboxylate (1.87 g, 5.0 mmol) was used as a raw material to obtain product Ik (0.20 g, 12% yield). The compound was analyzed using a nuclear magnetic resonance spectrometer to obtain 1 H NMR (600MHz, CDCl3) δ8.25(d,1H),8.36(d,1H),8.92(s,1H); 19 F NMR(565MHz, CDCl3)δ–61.22(s,3F).

[0102] Example 12: Preparation of 3-cyano-5-(2-furyl)-6-(trifluoromethyl)-1,2,4-triazine Il

[0103]

[0104] Using a method similar to Example 1, 6-(trifluoromethyl)-5-(2-furyl)-3-carboxylic acid methyl ester (1.37 g, 5.0 mmol) was used as a raw material to obtain product Il (0.96 g, 80% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ6.91(t,1H),7.71(d,1H),8.07(d,1H); 19 F NMR (565MHz, CDCl3) δ–62.01 (s, 3F).

[0105] Example 13: Preparation of 3,6-dicyano-5-phenyl-1,2,4-triazine II-a

[0106]

[0107] Take a 50mL round-bottom flask, add 5-phenyl-3,6-dicarboxylic acid methyl ester (1.37g, 5.0mmol) and 8mL methanol at room temperature, then add 25% concentrated ammonia (10mL) dropwise, react at room temperature for 6h, and the reaction is complete when detected by thin layer chromatography; remove methanol by thin film rotary evaporation, add 30ml of dichloromethane to the mixture, transfer to a separatory funnel for separation, wash the organic phase twice with saturated brine (10mL×2), dry with anhydrous sodium sulfate, and remove the solvent by thin film rotary evaporation to obtain the intermediate amide. Transfer it to a 25mL Schlenk bottle, dry it under high vacuum for 12 hours, replace it with nitrogen, then add anhydrous THF (8ml) and triethylamine (4.04g, 40.0mmol), place it at 0℃, add trifluoroacetic anhydride (3.16g, 15mmol) dropwise, and then return to room temperature for reaction for 12h. After the reaction was completed, 8 mL of water was added to quench the reaction; tetrahydrofuran was removed by thin film rotary evaporation, 30 mL of dichloromethane was added to the mixture, and the mixture was transferred to a separatory funnel for separation. The organic phase was washed twice with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed by thin film rotary evaporation. The crude product was separated by rapid silica gel column chromatography (first using 15:1 volume ratio of petroleum ether and ethyl acetate as elution solvent, and then using 5:1 volume ratio of petroleum ether and ethyl acetate as elution solvent) to obtain product II-a (0.73 g, 70% yield). The compound was analyzed by nuclear magnetic resonance, and the obtained 1 H NMR (600MHz, CDCl3) δ7.51(t,2H),7.64(t,1H),7.83(d,2H).

[0108] Example 14: Preparation of 3,6-dicyano-5-(4-methylphenyl)-1,2,4-triazine II-b

[0109]

[0110] Using a method similar to Example 13, 5-(4-methylphenyl)-3,6-dicarboxylic acid methyl ester (1.44 g, 5.0 mmol) was used as a raw material to obtain product II-b (0.82 g, 74% yield). The compound was analyzed using a nuclear magnetic resonance spectrometer to obtain 1 HNMR (600MHz, CDCl3) δ2.41(s,3H),7.33(d,2H),7.75(d,2H).

[0111] Example 15: Preparation of 3,6-dicyano-5-(4-methoxyphenyl)-1,2,4-triazine II-c

[0112]

[0113] Using a method similar to Example 13, 5-(4-methoxyphenyl)-3,6-dicarboxylic acid methyl ester (1.52 g, 5.0 mmol) was used as a raw material to obtain product II-c (0.77 g, 65% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ3.81(s,3H),7.10(d,2H),7.92(d,2H).

[0114] Example 16: Preparation of 3,6-dicyano-5-(4-fluorophenyl)-1,2,4-triazine II-d

[0115]

[0116] Using a method similar to Example 13, 5-(4-fluorophenyl)-3,6-dicarboxylic acid methyl ester (1.46 g, 5.0 mmol) was used as a raw material to obtain product II-d (0.56 g, 50% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 HNMR(600MHz, CDCl3)δ7.34(t,2H),7.83-7.87(m,2H); 19 F NMR (565MHz, CDCl3) δ – 105.77-105.84 (m, F).

[0117] Example 17: Preparation of 3,6-dicyano-5-(4-chlorophenyl)-1,2,4-triazine II-e

[0118]

[0119] A method similar to that of Experiment 13 was used, using 5-(4-chlorophenyl)-3,6-dicarboxylic acid methyl ester (1.54 g, 5.0 mmol) as the raw material to obtain product II-e (0.70 g, 58% yield). The compound was analyzed by nuclear magnetic resonance and obtained 1 H NMR (600MHz, CDCl3) δ7.46(d,2H),7.77(d,2H).

[0120] Example 18: Preparation of 3,6-dicyano-5-(4-bromophenyl)-1,2,4-triazine II-f

[0121]

[0122] A method similar to that of Experiment 13 was used, using 5-(4-bromophenyl)-3,6-dicarboxylic acid methyl ester (1.76 g, 5.0 mmol) as the raw material to obtain product II-f (1.03 g, 72% yield). The compound was analyzed by nuclear magnetic resonance and obtained1 H NMR (600MHz, CDCl3) δ7.58(d,2H),7.69(d,2H).

[0123] Example 19: Preparation of 3,6-dicyano-5-(4-iodophenyl)-1,2,4-triazine II-g

[0124]

[0125] Using a method similar to Example 13, 5-(4-iodophenyl)-3,6-dicarboxylic acid methyl ester (2.00 g, 5.0 mmol) was used as a raw material to obtain product II-g (1.10 g, 66% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 HNMR (600MHz, CDCl3) δ7.54(d,2H),7.92(d,2H).

[0126] Example 20: Preparation of 3,6-dicyano-5-[(4-trifluoromethyl)phenyl]-1,2,4-triazine II-h

[0127]

[0128] Using a method similar to Example 13, 5-[(4-trifluoromethyl)phenyl]-3,6-dicarboxylic acid methyl ester (1.71 g, 5.0 mmol) was used as a raw material to obtain product II-h (0.50 g, 36% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.81(d,2H),7.92(d,2H); 19 F NMR (565MHz, CDCl3) δ–63.23 (s, 3F).

[0129] Example 21: Preparation of 3,6-dicyano-5-(4-cyanophenyl)-1,2,4-triazine II-i

[0130]

[0131] Using a method similar to Example 13, 5-(4-cyanophenyl)-3,6-dicarboxylic acid methyl ester (1.49 g, 5.0 mmol) was used as the raw material to obtain product II-i (288 mg, 25% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.80(d,2H),7.88(d,2H).

[0132] Example 22: Preparation of 3,6-dicyano-5-(4-nitrophenyl)-1,2,4-triazine II-j

[0133]

[0134] Using a method similar to Example 13, 5-(4-nitrophenyl)-3,6-dicarboxylic acid methyl ester (1.59 g, 5.0 mmol) was used as a raw material to obtain product II-j (220 mg, 17% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ7.97(d,2H),8.51(d,2H).

[0135] Example 23: Preparation of 3,6-dicyano-5-(2,4-dinitrophenyl)-1,2,4-triazine II-k

[0136]

[0137] Using a method similar to Example 13, 5-(2,4-dinitrophenyl)-3,6-dicarboxylic acid methyl ester (1.82 g, 5.0 mmol) was used as a raw material to obtain product II-k (134 mg, 9% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600MHz, CDCl3) δ8.22(d,1H),8.31(d,1H),8.87(s,1H).

[0138] Example 24: Preparation of 3,6-dicyano-5-(2-furyl)-1,2,4-triazine II-1

[0139]

[0140] Using a method similar to Example 13, 5-(2-furyl)-3,6-dicarboxylic acid methyl ester (1.32 g, 5.0 mmol) was used as the raw material to obtain product II-1 (0.76 g, 77% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 HNMR (600MHz, CDCl3) δ6.84(t,1H),7.68(d,1H),8.01(d,1H).

[0141] Bioorthogonal ligation chemistry assay

[0142] The 3-cyano-5-aryl triazoxide obtained in Examples 1 to 24 was subjected to a one-pot cycloaddition / oxidation reaction with strained cyclooctenol (TCO and s-TCO), and the secondary kinetic rate constant was determined using an ultraviolet (UV)-stopped-flow spectrometer. 3-cyano-5-aryl triazoxide was prepared in acetonitrile / water (volume ratio 1 / 1) at four concentrations: 0.1 mM, 0.075 mM, 0.05 mM, and 0.025 mM. The molar ratio of strained cyclooctenol to 3-cyano-5-aryl triazoxide was 20:1. Each group of experiments was measured 3 times and the average value was taken to determine the secondary kinetic rate constant K2 (M -1 ·s -1 ) as shown in Table 1.

[0143] Table 1 Secondary kinetic rate constant detection results

[0144]

[0145]

[0146] The data in the above table show that 3-cyano-5-aryl triazine and strained cyclooctenol compounds can quickly undergo inverse electron demand Diels-Alder cycloaddition reaction (IED-DA), proving that such compounds can be used in bioorthogonal ligation chemical experiments and have great potential application value in chemical biology and bioimaging.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A 3-cyano-5-aryl-1,2,4-triazine compound, characterized in that: As shown in formula (I) or formula (II): Wherein, Ar is selected from substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-20 heterocyclic group; The substituents in the substituted C6-C20 aryl group and the substituted C3-C20 heterocyclic group are each independently selected from one or more of C1-C10 alkyl group, C1-C10 alkoxy group, C1-C10 haloalkyl group, halogen, cyano group and nitro group.

2. The 3-cyano-5-aryl-1,2,4-triazine compound according to claim 1, characterized in that Ar is selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C3-10 heterocyclic group; The substituents in the substituted C6-C10 aryl group and the substituted C3-C10 heterocyclic group are independently selected from one or more of C1-C5 alkyl group, C1-C5 alkoxy group, C1-C5 haloalkyl group, halogen, cyano group and nitro group.

3. The 3-cyano-5-aryl-1,2,4-triazine compound according to claim 1, characterized in that: Ar is selected from substituted or unsubstituted phenyl, substituted or unsubstituted furyl; The substituents in the substituted phenyl group and the substituted furyl group are independently selected from one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen, cyano and nitro.

4. The 3-cyano-5-aryl-1,2,4-triazine compound according to claim 1, characterized in that One of the structures shown in Ia~Il and II-a~II-l:

5. A method for preparing a 3-cyano-5-aryl-1,2,4-triazine compound, characterized in that: The following steps are involved: The compound represented by formula (IV) is subjected to a dehydration reaction under alkaline conditions using trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (I); Alternatively, the compound represented by formula (VI) is subjected to a dehydration reaction under alkaline conditions using trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound represented by formula (II); Wherein, Ar is selected from substituted or unsubstituted C6-C20 aryl, substituted or unsubstituted C3-20 heterocyclic group; The substituents in the substituted C6-C20 aryl group and the substituted C3-C20 heterocyclic group are each independently selected from one or more of C1-C10 alkyl group, C1-C10 alkoxy group, C1-C10 haloalkyl group, halogen, cyano group and nitro group.

6. The preparation method according to claim 5, characterized in that: The alkaline condition is provided by an organic base; the molar ratio of the trifluoroacetic anhydride to the organic base is 1:1 to 1:

3.

7. The preparation method according to claim 5, characterized in that: The compound represented by formula (IV) is prepared according to the following steps: The compound represented by formula (III) is reacted with an alcohol solution of aqueous ammonia to obtain a compound represented by formula (IV); And / or, the compound represented by formula (VI) is prepared according to the following steps: The compound represented by formula (V) is reacted with an alcohol solution of aqueous ammonia to obtain a compound represented by formula (VI); Wherein, R and R' are each independently selected from C1-C5 alkyl groups.

8. The preparation method according to claim 7, characterized in that: The mass concentration of the ammonia water is 25% to 38%; the volume ratio of the ammonia water to the alcohol solvent in the alcohol solution of the ammonia water is 1:0.5 to 1:

4.

9. Use of the 3-cyano-5-aryl-1,2,4-triazine compound described in any one of claims 1 to 4 and / or the 3-cyano-5-aryl-1,2,4-triazine compound prepared by the preparation method of any one of claims 5 to 8 as a small molecule probe.

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