3-Cyano-5-aryl-1,2,4-triazine compounds, their preparation methods and applications

Abstract

This 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 this invention can undergo a rapid one-pot cycloaddition / oxidation reaction with strained octenol compounds under room temperature and air conditions to generate the corresponding pyridine compounds, with a second-order rate constant K2 as high as 710 M. ‑1 ·s ‑1 This provides a novel small molecule probe for chemical biological research on bioorthogonal linkage reactions.

Description

TECHNICAL FIELD

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

[0002] It is of great significance to understand the molecular basis of life to specifically label biomacromolecules in vivo by bioorthogonal ligation of small molecule probes (E.M. Sletten, C.R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974-6998 and K. Liang, J.W. Chin, Chem. Rev. 2014, 114, 4764-4806). In recent years, J.A. Prescher in the United States, M.E. Webb in the United Kingdom and M. Vrabel in the Czech Republic have developed new bioorthogonal reactions by using 1,2,4-triazine as a small molecule probe and inverse electron-demand Diels-Alder cycloaddition (IED-DA) with strain trans-cyclooctene alcohols (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-triazines are either electron-rich or have a double aromatic substituent group at the 3,6-position of the ring, resulting in a slow cycloaddition rate with strain cyclooctene alcohols at room temperature (second-order rate constant K2 is between 0.075-9.9 M -1 ·s -1 ). Subsequently, the team of Ma Junan in China developed trifluoromethyl 1,2,4-triazine compounds, which greatly improved the cycloaddition rate with strain cyclooctene alcohols at room temperature (second-order rate constant K2 reached 99.2 M -1 ·s -1 )(J.-A. Ma, et al., ACS Catal. 2019, 9, 4600-4608). However, in order to improve the reaction efficiency, it is still urgent to design and synthesize new 1,2,4-triazine compounds for bioorthogonal ligation chemistry. SUMMARY

[0003] Therefore, the present application aims to provide a 3-cyano-5-aryl-1,2,4-triazine compound, a preparation method and application thereof, and the 3-cyano-5-aryl-1,2,4-triazine compound has a high cycloaddition reaction rate with a strain cyclooctene alcohol compound at room temperature.

[0004] The present application 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 and substituted or unsubstituted C3-C20 heterocyclyl;

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

[0008] Preferably, Ar is selected from substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C3-C10 heterocyclyl;

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

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

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

[0012] Preferably, as shown in one of structures I-a to I-l and II-a to II-l:

[0013]

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

[0015] dehydration of a compound shown in formula (IV) under alkaline conditions with trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (I);

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

[0017]

[0018] wherein Ar is selected from substituted or unsubstituted C6-C20 aryl and substituted or unsubstituted C3-C20 heterocyclyl;

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

[0020] Preferably, the basic conditions are provided by an organic base; and the molar ratio of trifluoroacetic anhydride to the organic base is 1:1-1:3.

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

[0022] reacting the compound shown as formula (III) with an alcoholic solution of ammonia to obtain the compound shown as formula (IV);

[0023] and / or, the compound shown as formula (VI) is prepared according to the following steps:

[0024] reacting the compound shown as formula (V) with an alcoholic solution of ammonia to obtain the compound shown as formula (VI);

[0025]

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

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

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

[0029] The present application provides a kind of 3-cyano-5-aryl-1,2,4-triazine compound, such as formula (I) or formula (II). Compared with prior art, the 3-cyano-5-aryl-1,2,4-triazine compound provided by the present application can be subjected to fast one-pot cycloaddition / oxidation reaction with tension cyclooctene alcohol compound under room temperature air condition, to generate corresponding pyridine compound, and the secondary rate constant K2 is as high as 710M -1 ·s -1 Provide a new small molecule probe for the chemical biology study of bioorthogonal ligation reaction. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The nuclear magnetic resonance hydrogen spectrum of compound I-f obtained in the embodiment 6 of the present application is shown in the following figure:

[0031] Figure 2 The nuclear magnetic resonance fluorine spectrum of compound I-f obtained in the embodiment 6 of the present application is shown in the following figure:

[0032] Figure 3 The nuclear magnetic resonance hydrogen spectrum of compound I-j obtained in the embodiment 10 of the present application is shown in the following figure:

[0033] Figure 4 The nuclear magnetic resonance fluorine spectrum of compound I-j obtained in the embodiment 10 of the present application is shown in the following figure. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0035] The present application provides a kind of 3-cyano-5-aryl-1,2,4-triazine compound, such as formula (I) or formula (II):

[0036]

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

[0038] In one embodiment, Ar is a substituted or unsubstituted C6-C15 aryl group or a substituted or unsubstituted C3-C15 heterocyclic group; each of the substituents in the substituted C6-C15 aryl group and the substituted C3-C15 heterocyclic group is 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, more preferably one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen, cyano, and nitro, and even more preferably one or more of methyl, ethyl, methoxy, ethoxy, halomethyl, haloethyl, halogen, cyano, and nitro; the halogen is any halogen known to one of ordinary skill in the art, and there is no particular limitation; and the heteroatom in the heterocyclic group is any heteroatom known to one of ordinary skill in the art, and there is no particular limitation.

[0039] In one embodiment, Ar is a substituted or unsubstituted C6-C10 aryl group or a substituted or unsubstituted C3-C10 heterocyclic group; each of the substituents in the substituted C6-C10 aryl group and the substituted C3-C10 heterocyclic group is 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, more preferably one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen, cyano, and nitro, and even more preferably one or more of methyl, ethyl, methoxy, ethoxy, halomethyl, haloethyl, halogen, cyano, and nitro; the halogen is any halogen known to one of ordinary skill in the art, and there is no particular limitation; and the heteroatom in the heterocyclic group is any heteroatom known to one of ordinary skill in the art, and there is no particular limitation.

[0040] In one specific embodiment provided by the present application, Ar is substituted or unsubstituted phenyl, substituted or unsubstituted furanyl; the substituents in the substituted phenyl and the substituted furanyl 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, more preferably one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen, cyano and nitro, and even more preferably one or more of methyl, ethyl, methoxy, ethoxy, halomethyl, haloethyl, halogen, cyano and nitro; the halogen can be any halogen known to those skilled in the art without any particular limitation, and in the present application, one or more of fluorine, chlorine and bromine is preferred; the heteroatom in the heterocyclyl group can be any heteroatom known to those skilled in the art without any particular limitation, and in the present application, nitrogen, oxygen or sulfur is preferred.

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

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

[0043]

[0044] The 3-cyano-5-aryl-1,2,4-triazine compound provided by the present application can be subjected to a fast one-pot cycloaddition / oxidation reaction with a strain cyclooctene alcohol compound under room temperature air conditions to generate a corresponding pyridine compound, and the secondary rate constant K2 is as high as 710 M -1 ·s -1 , which provides a novel small-molecule probe for the biochemical study of bioorthogonal ligation reactions.

[0045] The present application also provides a preparation method of the above-mentioned 3-cyano-5-aryl-1,2,4-triazine compound, which comprises the following steps: subjecting a compound shown in formula (IV) 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 shown in formula (I);

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

[0047]

[0048] wherein Ar is a substituted or unsubstituted C6-C20 aryl group or 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, C1-C10 alkoxy, C1-C10 haloalkyl, halogen, cyano and nitro.

[0049] The present application does not have special limitation on the source of all raw materials, which are commercially available; the Ar is as described above, which is not repeated here.

[0050] In a specific embodiment provided by the present application, the compound shown as formula (IV) is prepared by the following steps: reacting the compound shown as formula (III) with an alcohol solution of ammonia to obtain the compound shown as formula (IV); the mass concentration of the ammonia is preferably 25%-38%, more preferably 25%-35%; the volume ratio of ammonia to alcohol solvent in the alcohol solution of ammonia is preferably 1:0.5-1:4, more preferably 1:0.5-1:3, again preferably 1:0.5-1:2, again preferably 1:0.5-1:1.5, most preferably 1:0.8-1:1; the alcohol solvent can be any alcohol solvent known to those skilled in the art without special limitation, which is preferably methanol in the present application; in the present application, the compound shown as formula (III) is preferably mixed with the alcohol solvent first, and then ammonia is added dropwise; the reaction is preferably carried out at room temperature; the reaction time is preferably 4-10h, more preferably 4-8h, again preferably 6h; after the reaction is completed, the alcohol solvent is preferably removed, then dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a drying agent, and then the solvent is removed to obtain the compound shown as formula (IV); the method for removing the alcohol solvent and the method for removing the solvent can be any method known to those skilled in the art without special limitation, which is preferably thin film rotary evaporation in the present application; the saturated brine washing is preferably carried out 2-3 times; the drying agent is preferably anhydrous sodium sulfate.

[0051]

[0052] wherein R is C1-C5 alkyl, preferably C1-C3 alkyl, more preferably methyl or ethyl.

[0053] The compound shown in formula (IV) is dehydrated under alkaline condition with trifluoroacetic anhydride (TFAA) as a dehydrating agent to obtain the 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (I); in the present application, the compound shown in formula (IV) is preferably dried before dehydrated under alkaline condition with trifluoroacetic anhydride (TFAA) as a dehydrating agent; the drying is preferably high vacuum drying; the time of the high vacuum drying is preferably 8-20h, more preferably 10-15h, and even more preferably 12h; the alkaline condition is preferably provided by an organic base; the organic base is any organic base known to those skilled in the art without special limitation, and in the present application, triethylamine (TEA) is preferred; the molar ratio of trifluoroacetic anhydride to the organic base is preferably 1:1-1:3, more preferably 1:1.5-1:3, even more preferably 1:2-1:3, and most preferably 1:2.5-1:2.7; the dehydrating reaction is preferably carried out in an organic solvent; the organic solvent is any organic solvent known to those skilled in the art without special limitation, and in the present application, tetrahydrofuran is preferred; in the present application, the compound shown in formula (IV) is preferably mixed with an organic solvent and an organic base first, and then trifluoroacetic anhydride is added under low temperature condition, and the dehydrating reaction is carried out by warming; the mixing and the dehydrating reaction are preferably carried out in a protective atmosphere; the protective atmosphere is any protective atmosphere known to those skilled in the art without special limitation, and in the present application, nitrogen is preferred; the temperature of the low temperature condition is preferably 0-3℃; the dehydrating reaction is preferably carried out at room temperature; the time of the dehydrating reaction is preferably 8-20h, more preferably 10-15h, and even more preferably 12h; after the dehydrating reaction is completed, water is preferably added to quench the reaction, and after the organic solvent is removed, dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a drying agent, and then the solvent is removed, and after purification, the 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (I) is obtained; the method of removing the organic solvent and removing the solvent is any method known to those skilled in the art without special limitation, and in the present application, thin film rotary evaporation is preferred; the number of times of washing with saturated brine is preferably 2-3 times; the drying agent is preferably anhydrous sodium sulfate; the purification is preferably silica gel column chromatography, and more preferably flash silica gel column chromatography; the elution solvent used in the purification is preferably petroleum ether and ethyl acetate; in the present application, petroleum ether and ethyl acetate with a volume ratio of 20:1 are used as eluent first, and then petroleum ether and ethyl acetate with a volume ratio of 10:1 are used as eluent.

[0054] In a specific embodiment provided in the present application, the 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (I) is prepared according to the following reaction formula:

[0055]

[0056] In a specific embodiment provided by the present application, the compound shown as formula (VI) is prepared by the following steps: reacting the compound shown as formula (V) with an alcohol solution of ammonia to obtain the compound shown as formula (VI); the mass concentration of the ammonia is preferably 25% to 38%, more preferably 25% to 35%; the volume ratio of ammonia to alcohol solvent in the alcohol solution of ammonia 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 can be any alcohol solvent known to those skilled in the art without special limitation, and is preferably methanol in the present application; in the present application, the compound shown as formula (V) is preferably mixed with the alcohol solvent first, and then ammonia is added 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 is completed, the alcohol solvent is removed, then dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a drying agent, and then the solvent is removed to obtain the compound shown as formula (VI); the number of times of washing with saturated brine is preferably 2 to 3 times; and the drying agent is preferably anhydrous sodium sulfate.

[0057]

[0058] wherein R' is C1-C5 alkyl, preferably C1-C3 alkyl, and more preferably methyl or ethyl.

[0059] The compound shown in formula (VI) is dehydrated under alkaline condition with trifluoroacetic anhydride as a dehydrating agent to obtain a 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (II); in the present application, the compound shown in formula (VI) is preferably dried before dehydrated under alkaline condition with trifluoroacetic anhydride (TFAA) as a dehydrating agent; the drying is preferably high vacuum drying; the time of the high vacuum drying is preferably 8-20h, more preferably 10-15h, and even more preferably 12h; the alkaline condition is preferably provided by an organic base; the organic base is any organic base known to those skilled in the art without special limitation, and in the present application, triethylamine (TEA) is preferred; the molar ratio of trifluoroacetic anhydride to the organic base is preferably 1:1-1:3, more preferably 1:1.5-1:3, even more preferably 1:2-1:3, and most preferably 1:2.5-1:2.7; the dehydrating reaction is preferably carried out in an organic solvent; the organic solvent is any organic solvent known to those skilled in the art without special limitation, and in the present application, tetrahydrofuran is preferred; in the present application, the compound shown in formula (VI) is preferably mixed with an organic solvent and an organic base first, and then trifluoroacetic anhydride is added under low temperature condition, and the dehydrating reaction is carried out by warming; the mixing and the dehydrating reaction are preferably carried out in a protective atmosphere; the protective atmosphere is any protective atmosphere known to those skilled in the art without special limitation, and in the present application, nitrogen is preferred; the temperature of the low temperature condition is preferably 0-3℃; the dehydrating reaction is preferably carried out at room temperature; the time of the dehydrating reaction is preferably 8-20h, more preferably 10-15h, and even more preferably 12h; after the dehydrating reaction is completed, water is preferably added to quench the reaction, and after the organic solvent is removed, dichloromethane is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a drying agent, and then the solvent is removed, and after purification, a 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (II) is obtained; the method of removing the organic solvent and removing the solvent is any method known to those skilled in the art without special limitation, and in the present application, thin film rotary evaporation is preferred; the number of times of washing with saturated brine is preferably 2-3 times; the drying agent is preferably anhydrous sodium sulfate; the purification is preferably silica gel column chromatography, and more preferably flash silica gel column chromatography; the elution solvent used in the purification is preferably petroleum ether and ethyl acetate; in the present application, petroleum ether and ethyl acetate with a volume ratio of 15:1 are used as eluent first, and then petroleum ether and ethyl acetate with a volume ratio of 5:1 are used as eluent.

[0060] In a specific embodiment provided in the present application, the 3-cyano-5-aryl-1,2,4-triazine compound shown in formula (II) is prepared according to the following reaction formula:

[0061]

[0062] The application further provides application of the 3-cyano-5-aryl-1,2,4-triazine compound as a small molecule probe.

[0063] In a specific embodiment provided by the application, the 3-cyano-5-aryl-1,2,4-triazine compound can quickly perform a cycloaddition reaction with a strained cyclooctyne compound to generate a pyridine compound, and thus the 3-cyano-5-aryl-1,2,4-triazine compound can be used as a small molecule probe for biological ortho-linking reaction.

[0064] Further, the cycloaddition reaction is performed at room temperature and in air.

[0065] In order to further illustrate the application, the following embodiments are used to describe the 3-cyano-5-aryl-1,2,4-triazine compound, the preparation method and the application thereof in detail.

[0066] The reagents used in the following examples are commercially available. The standard silica gel with a particle size of 200-300 mesh is used for flash column chromatography, and the 0.20 mm standard plate is used for thin layer chromatography. The nuclear magnetic resonance spectrum data (NMR) is obtained by using a Bruker 400 megahertz nuclear magnetic resonance instrument, with tetramethylsilane as an internal standard and deuterated chloroform as a solvent (s represents a single peak, 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 I-a

[0068]

[0069] Into a 25 mL round bottom flask, 6-(trifluoromethyl)-5-phenyl-3-methyl carboxylate (1.42 g, 5.0 mmol) and 5 mL of methanol were added at room temperature, followed by dropwise addition of 25% concentrated aqueous ammonia (5 mL) at room temperature. The reaction was allowed to proceed for 6 h, and thin layer chromatography was used to determine that the reaction was complete. Methanol was removed by rotary evaporation, and 30 mL of dichloromethane was added to the mixture. The mixture was transferred to a separatory funnel, and the organic phase was washed twice with saturated aqueous sodium chloride (10 mL x 2), dried over anhydrous sodium sulfate, and rotary evaporated to remove the solvent to obtain the intermediate amide. The intermediate amide was transferred to a 25 mL Schlenk flask, dried under high vacuum for 12 h, and then replaced with nitrogen. Anhydrous THF (8 mL) and triethylamine (2.02 g, 20.0 mmol) were added, and the mixture was cooled to 0 °C. Trifluoroacetic anhydride (1.58 g, 7.5 mmol) was added dropwise, and the mixture was allowed to warm to room temperature and react for 12 h. After the reaction was completed, 5 mL of water was added to quench the reaction. The solvent was removed by rotary evaporation, and 30 mL of dichloromethane was added to the mixture. The mixture was transferred to a separatory funnel, and the organic phase was washed twice with saturated aqueous sodium chloride (10 mL x 2), dried over anhydrous sodium sulfate, and rotary evaporated to remove the solvent. The crude product was purified by flash column chromatography using silica gel (first with 20: 1 volume ratio of petroleum ether and ethyl acetate as the eluent, and then with 10: 1 volume ratio of petroleum ether and ethyl acetate as the eluent) to obtain the product I-a (0.94 g, 75% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600 MHz, CDC13) δ 7.49-7.53 (m, 3H), 7.72 (d, 2H); 19 F NMR (565 MHz, CDC13) δ -61.30 (s, 3F).

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

[0071]

[0072] The product I-b (1.03 g, 78% yield) was obtained using a method similar to that of Example 1, starting from 6-(trifluoromethyl)-5-(4-methylphenyl)-3-methyl carboxylate (1.49 g, 5.0 mmol). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600 MHz, CDC13) δ 2.33 (s, 3H), 7.37 (d, 2H), 7.76 (d, 2H); 19 F NMR (565 MHz, CDC13) δ -61.19 (s, 3F).

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

[0074]

[0075] Using a similar procedure as in Example 1, starting with 6-(trifluoromethyl)-5-(4- methoxyphenyl)-3-methyl carboxylate (1.57 g, 5.0 mmol), the product I-c (1.13 g, 81% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 3.83 (s, 3H), 7.04 (d, 2H), 7.85 (d, 2H); 19 F NMR (565 MHz, CDC13) δ -61.22 (s, 3F).

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

[0077]

[0078] Using a similar procedure as in Example 1, starting with 6-(trifluoromethyl)-5-(4- methoxyphenyl)-3-methyl carboxylate (1.57 g, 5.0 mmol), the product I-c (1.13 g, 81% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 3.83 (s, 3H), 7.04 (d, 2H), 7.85 (d, 2H); 19 F NMR (565 MHz, CDC13) δ -61.22 (s, 3F).

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

[0080]

[0081] Using a similar procedure as in Example 1, starting with 6-(trifluoromethyl)-5-(4- methoxyphenyl)-3-methyl carboxylate (1.57 g, 5.0 mmol), the product I-c (1.13 g, 81% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 3.83 (s, 3H), 7.04 (d, 2H), 7.85 (d, 2H); 19F NMR (565 MHz, CDCI3) δ -61.00 (s, 3F).

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

[0083]

[0084] Using a similar method to Example 1, starting with 6-(trifluoromethyl)-5-(4- bromophenyl)-3-methyl formate (1.81 g, 5.0 mmol), the product I-f (0.95 g, 58% yield) was obtained.

[0085] The compound was analyzed using a nuclear magnetic resonance spectrometer to obtain a hydrogen nuclear magnetic resonance spectrum as shown in Figure 1 The results are shown in Table 1 below. 1 H NMR (600 MHz, CDCI3) δ 7.77 (d, 2H), 7.71 (d, 2H); a fluorine nuclear magnetic resonance spectrum as shown in Figure 2 The results are shown in Table 1 below. 19 F NMR (565 MHz, CDCI3) δ -61.00 (s, 3F).

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

[0087]

[0088] Using a similar method to Example 1, starting with 6-(trifluoromethyl)-5-(4- bromophenyl)-3-methyl formate (1.81 g, 5.0 mmol), the product I-f (0.95 g, 58% yield) was obtained. 1 H NMR (600 MHz, CDCI3) δ 7.60-7.73 (m, 4H); 19 F NMR (565 MHz, CDCI3) δ -61.00 (s, 3F).

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

[0090]

[0091] Using a similar method to Example 1, starting from 6-(trifluoromethyl)-5-[(4- trifluoromethyl)phenyl]-3-methyl formate (1.76 g, 5.0 mmol), the product I-h (0.65 g, 41% yield) was obtained. The compound was analyzed by NMR to give the NMR hydrogen spectrum as shown in 1 H NMR (600 MHz, CDC13) δ 7.83 (d, 2H), 7.95 (d, 2H); 19 F NMR (565 MHz, CDC13) δ -61.21 (s, 3F), -63.23 (s, 3F).

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

[0093]

[0094] Using a similar method to Example 1, starting from 6-(trifluoromethyl)-5-(4- cyanophenyl)-3-methyl formate (1.54 g, 5.0 mmol), the product I-i (0.36 g, 26% yield) was obtained. The compound was analyzed by NMR to give the NMR hydrogen spectrum as shown in 1 H NMR (600 MHz, CDC13) δ 7.89 (dd, 4H); 19 F NMR (565 MHz, CDC13) δ -61.17 (s, 3F).

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

[0096]

[0097] Using a similar method to Example 1, starting from 6-(trifluoromethyl)-5-(4- nitrophenyl)-3-methyl formate (1.64 g, 5.0 mmol), the product I-j (0.27 g, 18% yield) was obtained.

[0098] The compound was analyzed by NMR to give the NMR hydrogen spectrum as shown in Figure 3 and the results were 1 H NMR (600 MHz, CDC13) δ 8.49 (d, 2H), 8.01 (d, 2H); the NMR fluorine spectrum as shown in Figure 4 and the results were 19 F NMR (565 MHz, CDC13) δ -61.21 (s, 3F).

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

[0100]

[0101] Using a similar procedure to Example 1, starting with 6-(trifluoromethyl)-5-(2,4- dinitrophenyl)-3-methyl formate (1.87 g, 5.0 mmol), the product I-k (0.20 g, 12% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 8.25 (d, 1H), 8.36 (d, 1H), 8.92 (s, 1H); 19 F NMR (565 MHz, CDC13) δ -61.22 (s, 3F).

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

[0103]

[0104] Using a similar procedure to Example 1, starting with 6-(trifluoromethyl)-5-(2,4- dinitrophenyl)-3-methyl formate (1.87 g, 5.0 mmol), the product I-k (0.20 g, 12% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 8.25 (d, 1H), 8.36 (d, 1H), 8.92 (s, 1H); 19 F NMR (565 MHz, CDC13) δ -61.22 (s, 3F).

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

[0106]

[0107] Into a 50 mL round bottom flask, 5-phenyl-3,6-bismethyl carboxylate (1.37 g, 5.0 mmol) and 8 mL of methanol were added at room temperature, followed by dropwise addition of 25% concentrated aqueous ammonia (10 mL) at room temperature. The reaction was allowed to proceed for 6 h, and thin layer chromatography was used to determine completion of the reaction. Methanol was removed by rotary evaporation, and 30 mL of dichloromethane was added to the mixture, which was then transferred to a separatory funnel. The organic phase was washed twice with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the intermediate amide. The intermediate amide was transferred to a 25 mL Schlenk flask, dried under high vacuum for 12 h, and then replaced with nitrogen. Anhydrous THF (8 mL) and triethylamine (4.04 g, 40.0 mmol) were added, and the mixture was cooled to 0 °C. Trifluoroacetic anhydride (3.16 g, 15 mmol) was added dropwise, and the reaction was allowed to proceed at room temperature for 12 h. After the reaction was completed, 8 mL of water was added to quench the reaction. Tetrahydrofuran was removed by rotary evaporation, and 30 mL of dichloromethane was added to the mixture, which was then transferred to a separatory funnel. The organic phase was washed twice with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by flash column chromatography using silica gel (first with 15: 1 volume ratio of petroleum ether and ethyl acetate, and then with 5: 1 volume ratio of petroleum ether and ethyl acetate) to obtain the product II-a (0.73 g, 70% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600 MHz, CDC13) δ 7.51 (t, 2H), 7.64 (t, 1H), 7.83 (d, 2H).

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

[0109]

[0110] Using a similar method to that of Example 13, 5-(4-methylphenyl)-3,6-bismethyl carboxylate (1.44 g, 5.0 mmol) was used as the starting material to obtain the product II-b (0.82 g, 74% yield). The compound was analyzed by nuclear magnetic resonance to obtain 1 H NMR (600 MHz, CDC13) δ 7.51 (t, 2H), 7.64 (t, 1H), 7.83 (d, 2H).

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

[0112]

[0113] The product II-c (0.77 g, 65% yield) was obtained using a similar procedure as in Example 13, starting from 5-(4-methoxyphenyl)-3,6-biscarboxylic acid methyl ester (1.52 g, 5.0 mmol). The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 3.81 (s, 3H), 7.10 (d, 2H), 7.92 (d, 2H).

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

[0115]

[0116] The product II-d (0.56 g, 50% yield) was obtained using a similar procedure as in Example 13, starting from 5-(4-fluorophenyl)-3,6-biscarboxylic acid methyl ester (1.46 g, 5.0 mmol). The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 7.34 (t, 2H), 7.83-7.87 (m, 2H); 19 F NMR (565 MHz, CDC13) δ -105.77-105.84 (m, F).

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

[0118]

[0119] The product II-e (0.70 g, 58% yield) was obtained using a similar procedure as in Example 13, starting from 5-(4-chlorophenyl)-3,6-biscarboxylic acid methyl ester (1.54 g, 5.0 mmol). The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 7.46 (d, 2H), 7.77 (d, 2H).

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

[0121]

[0122] The product II-f (1.03 g, 72% yield) was obtained using a similar procedure as in Example 13, starting from 5-(4-bromophenyl)-3,6-biscarboxylic acid methyl ester (1.76 g, 5.0 mmol). The compound was analyzed by NMR to give1 H NMR (600 MHz, CDC13) δ 7.58 (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 similar procedure as in Example 13, starting with 5-(4-iodophenyl)-3,6- dimethyl ester (2.00 g, 5.0 mmol), the product II-g (1.10 g, 66% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 7.54 (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 similar procedure as in Example 13, starting with 5-[(4-trifluoromethyl)phenyl]- 3,6-dimethyl ester (1.71 g, 5.0 mmol), the product II-h (0.50 g, 36% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 7.81 (d, 2H), 7.92 (d, 2H); 19 F NMR (565 MHz, CDC13) 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 similar procedure as in Example 13, starting with 5-(4-cyanophenyl)-3,6- dimethyl ester (1.49 g, 5.0 mmol), the product II-i (288 mg, 25% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 7.80 (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 similar procedure as in Example 13, starting with 5-(4-nitrophenyl)-3,6- dimethyl ester (1.59 g, 5.0 mmol), the product II-j (220 mg, 17% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 7.97 (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 similar procedure as in Example 13, starting with 5-(2,4-dinitrophenyl)-3,6- dimethyl ester (1.82 g, 5.0 mmol), the product II-k (134 mg, 9% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 8.22 (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-l

[0139]

[0140] Using a similar procedure as in Example 13, starting with 5-(2-furyl)-3,6-dimethyl ester (1.32 g, 5.0 mmol), the product II-l (0.76 g, 77% yield) was obtained. The compound was analyzed by NMR to give 1 H NMR (600 MHz, CDC13) δ 6.84 (t, 1H), 7.68 (d, 1H), 8.01 (d, 1H).

[0141] Bioorthogonal ligation chemistry experiments

[0142] One-pot cycloaddition / oxidation reaction of 3-cyano-5-aryltriazazines obtained in Example 1 to Example 24 with Tensioned Cyclooctene alcohols (TCO and s-TCO) was carried out, and the second-order kinetic rate constant was determined by UV-stopped-flow device. 3-cyano-5-aryltriazazines were prepared in acetonitrile / water (volume ratio 1 / 1) at four concentrations: 0.1 mM, 0.075 mM, 0.05 mM, 0.025 mM, and the molar ratio of Tensioned Cyclooctene alcohols to 3-cyano-5-aryltriazazines was 20:1 in each experiment, and the second-order kinetic rate constant K2 (M-1s-1) was determined by UV-stopped-flow device, and the average value of three experiments was taken. -1 ·s -1 ) as shown in Table 1.

[0143] Table 1: Second-order kinetic rate constant detection results

[0144]

[0145]

[0146] The data in the above table shows that 3-cyano-5-aryltriazazines can rapidly undergo inverse electron demand Diels-Alder cycloaddition (IED-DA) with Tensioned Cyclooctene alcohols, proving that these compounds can be used for biological orthogonal ligation chemistry experiments, and have great potential application value in chemical biology and bioimaging.

[0147] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A 3-cyano-5-aryl-l,2,4-triazine compound, characterized by, As shown in formula (I) or formula (II): wherein Ar is selected from substituted or unsubstituted phenyl, substituted or unsubstituted furanyl; each of the substituents in the substituted phenyl and the substituted furanyl is independently one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen, cyano, and nitro.

2. The 3-cyano-5-aryl-l,2,4-triazine compound according to claim 1, characterized by The substituents in the substituted phenyl and the substituted furanyl are each independently selected from one or more of C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, halogen, cyano and nitro.

3. The 3-cyano-5-aryl-l,2,4-triazine compound according to claim 1, characterized by One of the structures shown in I-a-I-l and II-a-II-l: 。 4. A process for the preparation of a 3-cyano-5-aryl-l,2,4-triazine compound, characterized in that, The method comprises the following steps: The compound shown in formula (IV) 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 shown in formula (I); Alternatively, the compound shown in 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 shown in formula (II); wherein said Ar is selected from substituted or unsubstituted phenyl, substituted or unsubstituted furanyl; The substituents in the substituted phenyl and the substituted furanyl are each independently selected from one or more of C1-C10 alkyl, C1-C10 alkoxy, C1-C10 haloalkyl, halogen, cyano and nitro.

5. The preparation method according to claim 4, characterized in that, The alkaline conditions are provided by an organic base; and the molar ratio of the trifluoroacetic anhydride to the organic base is 1:1-1:

3.

6. The preparation method according to claim 4, characterized in that, The compound shown in formula (IV) is prepared according to the following steps: The compound shown in formula (III) is reacted with an alcohol solution of ammonia water to obtain the compound shown in formula (IV); And / or, the compound shown in formula (VI) is prepared according to the following steps: The compound shown in formula (V) is reacted with an alcohol solution of ammonia water to obtain the compound shown in formula (VI); wherein R and R' are each independently selected from C1-C5 alkyl groups.

7. The preparation method according to claim 5, characterized in that, The mass concentration of the ammonia water is 25%-38%; and the volume ratio of the ammonia water to the alcohol solvent in the alcohol solution of ammonia water is 1:0.5-1:

4.

8. Use of the 3-cyano-5-aryl-1,2,4-triazine compound according to any one of claims 1-3 and / or the 3-cyano-5-aryl-1,2,4-triazine compound prepared by the method according to any one of claims 4-7 in the preparation of a small-molecule probe for a biological orthogonal ligation reaction; the biological orthogonal ligation reaction is a cycloaddition reaction of the 3-cyano-5-aryl-1,2,4-triazine compound as a diene donor with a strain cyclooctene alcohol compound to generate a pyridine compound.

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