Non-natural chiral amino acid containing cyano triazazine as well as preparation method and application of non-natural chiral amino acid

Abstract

The invention provides a non-natural chiral amino acid containing cyano triazazine or a pharmaceutically acceptable salt thereof. The non-natural chiral amino acid containing cyano triazazine or the pharmaceutically acceptable salt thereof comprises a structure as shown in a formula (I). Compared with the prior art, the non-natural chiral amino acid containing cyano triazine or the pharmaceutically acceptable salt thereof provided by the invention can be subjected to a one-pot cycloaddition / oxidation reaction with tension cyclooctenol compounds under a room-temperature air condition to quickly generate corresponding pyridine compounds, the secondary rate constant K2 can be up to 975M <-1 >. S <-1 >, and the chiral amino acid or pharmaceutically acceptable salt thereof can be used as a novel chiral compound. The novel non-natural chiral alpha-amino acid is provided for the biological orthogonal link reaction, and can be further applied to chemical and biological research based on the biological orthogonal link reaction. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of non-natural chiral amino acids, and in particular relates to a non-natural chiral amino acid containing cyanotriazoxide, and a preparation method and application thereof. Background Art

[0002] Amino acids are the basic substances that constitute life, and their synthesis and application have also attracted extensive attention from scientific researchers. Proteins in living organisms are composed of a limited combination of 20 natural chiral α-amino acids, which greatly limits the application scope of protein chemistry and function. In recent years, the gene codon expansion (GCE) technology has achieved the introduction of unnatural amino acids at specific sites of any target protein in the body. This technology uses aminoacyl-tRNA synthetase / tRNA pairs (aaRS / tRNA pairs) that are orthogonal to the host cell, and encodes unnatural amino acids through stop codons (UAG / UGA / UAA) (Peter G. Schultz et al., Annu. Rev. Biochem. 2010, 79, 413–44). To date, more than 200 non-natural chiral α-amino acids have been site-specifically introduced into peptides or proteins through genetic codon expansion technology; and all non-natural chiral α-amino acids with bioorthogonal functional groups are derived from natural aromatic amino acids (such as phenylalanine, tryptophan, tyrosine and histidine), serine, threonine, (cysteine) and lysine (Tao Liu et al., Angew. Chem. Int. Ed. 2021, 60, 10040–10048; Nediljko Budisa et al., Angew. Chem. Int. Ed. 2017, 56, 9680–9703; & Benjamin G. Davis et al., Chem. Sci. 2015, 6, 50–69).

[0003] In recent years, researchers have tried to use the inverse electron-demand Diels-Alder cycloaddition (IED-DA) of 1,2,4-triazine and strained cyclooctenol compounds to develop new bioorthogonal reactions (JA Prescher, et al., J. Am. Chem. Soc. 2015, 137, 8388-8391; ME Webb, et al., Chem. Eur. J. 2015, 21, 14376–14381; M. Vrabel, et al., Chem. Sci. 2017, 8, 3593–3598; & J.-A. Ma, et al., ACS Catal. 2019, 9, 4600–4608). However, in the reverse electron-demanding Diels-Alder cycloaddition reaction of these 1,2,4-triazine compounds with strained cyclooctenols, the highest secondary rate constant K2 is only 100 M -1 ·s -1 Therefore, it is urgent to design and synthesize unnatural chiral amino acids containing novel triazoxide heterocycles to improve their reaction efficiency. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a non-natural chiral amino acid containing cyanotriazoxide, a preparation method and application thereof, wherein the non-natural chiral amino acid containing cyanotriazoxide has a high cycloaddition reaction rate with a strained cyclooctenol compound under room temperature conditions.

[0005] The present invention provides a non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, comprising a structure shown in formula (I):

[0006]

[0007] Wherein, n is an integer from 0 to 5;

[0008] R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro;

[0009] X is selected from heteroatoms, C1-C10 alkylene groups;

[0010] The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

[0011] Preferably, n is 0 or 1.

[0012] Preferably, the R is selected from substituted or unsubstituted C1-C5 alkyl, cyano or nitro; the substituent of the substituted C1-C5 alkyl is selected from one or more of halogen, cyano and nitro.

[0013] Preferably, said R is selected from trifluoromethyl, cyano or nitro;

[0014] And / or, X is selected from oxygen, sulfur, selenium, and C1-C5 alkylene.

[0015] Preferably, it includes one of the structures shown in Ia~Ij:

[0016]

[0017] Preferably, the pharmaceutically acceptable salt is trifluoroacetate or hydrochloride.

[0018] The present invention also provides a method for preparing a non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0019] S1) reacting the compound represented by formula (II) with the compound represented by formula (III) in the presence of an acid-binding agent to obtain a non-natural chiral amino acid ester represented by formula (IV);

[0020] S2) removing the protecting group from the non-natural chiral amino acid ester represented by formula (IV) to obtain the non-natural chiral amino acid containing cyanotriazoxide represented by formula (I) or a pharmaceutically acceptable salt thereof;

[0021]

[0022] Wherein, n is an integer from 0 to 5;

[0023] R1 is an amino protecting group; R2 is a carboxyl protecting group;

[0024] R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro;

[0025] X is selected from heteroatoms, C1-C10 alkylene groups;

[0026] The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

[0027] Preferably, R1 is tert-butyloxycarbonyl; R2 is tert-butyl;

[0028] The acid binding agent is selected from one or more of triethylamine, N,N-diisopropylethylamine, N-methylmorpholine and 4-dimethylaminopyridine.

[0029] The present invention also provides a non-natural chiral amino acid ester containing cyanotriazoxide, as shown in formula (IV):

[0030]

[0031] Wherein, n is an integer from 0 to 5;

[0032] R1 is an amino protecting group; R2 is a carboxyl protecting group;

[0033] R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro;

[0034] X is selected from heteroatoms, C1-C10 alkylene groups;

[0035] The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

[0036] The present invention also provides a use of the non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof or the non-natural chiral amino acid ester containing cyanotriazoxide as a small molecule probe.

[0037] The present invention provides a non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, including a structure shown in formula (I). Compared with the prior art, the non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof provided by the present invention can undergo a one-pot cycloaddition / oxidation reaction with a strained cyclooctenol compound under room temperature and air conditions to quickly generate a corresponding pyridine compound, and the secondary rate constant K2 can reach up to 975M -1 ·s -1 , providing a new class of non-natural chiral α-amino acids for bioorthogonal linking reactions, which can be further applied to chemical biology research based on bioorthogonal linking reactions. DETAILED DESCRIPTION

[0038] 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.

[0039] The present invention provides a non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, comprising a structure shown in formula (I):

[0040]

[0041] Wherein, n is an integer from 0 to 5;

[0042] R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro;

[0043] X is selected from heteroatoms, C1-C10 alkylene groups;

[0044] The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

[0045] In a specific embodiment provided by the present invention, the n is preferably an integer of 0 to 3, more preferably 0, 1 or 2, and even more preferably 0 or 1.

[0046] In a specific embodiment provided by the present invention, the R is preferably a substituted or unsubstituted C1-C5 alkyl, cyano or nitro; the substituent of the substituted C1-C5 alkyl is preferably one or more of halogen, cyano and nitro; the halogen is any halogen well known to those skilled in the art without any special limitation, and is preferably fluorine, chlorine or bromine in the present invention.

[0047] In a specific embodiment provided by the present invention, the R is preferably a substituted or unsubstituted C1-C3 alkyl, cyano or nitro; the substituent of the substituted C1-C3 alkyl is preferably one or more of halogen, cyano and nitro; the halogen is any halogen well known to those skilled in the art without any special limitation, and is preferably fluorine, chlorine or bromine in the present invention.

[0048] In a specific embodiment provided by the present invention, the R is preferably a substituted or unsubstituted C1-C2 alkyl, cyano or nitro; the substituent of the substituted C1-C2 alkyl is preferably one or more of halogen, cyano and nitro; the halogen is any halogen well known to those skilled in the art without any special limitation, and in the present invention, it is preferably fluorine, chlorine or bromine.

[0049] In a specific embodiment provided by the present invention, the R is preferably trifluoromethyl, cyano or nitro.

[0050] In a specific embodiment provided by the present invention, X is preferably oxygen, sulfur, selenium, or a C1-C5 alkylene group, more preferably oxygen, sulfur, selenium, or a C1-C3 alkylene group, and even more preferably oxygen, sulfur, selenium, or a methylene group.

[0051] In a specific embodiment provided by the present invention, the non-natural chiral amino acid or a pharmaceutically acceptable salt thereof includes one of the structures shown in Ia to Ij:

[0052]

[0053] In a specific embodiment provided by the present invention, the pharmaceutically acceptable salt is trifluoroacetate or hydrochloride.

[0054] The present invention also provides a method for preparing the above-mentioned non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, comprising the following steps: S1) reacting a compound represented by formula (II) with a compound represented by formula (III) in the presence of an acid-binding agent to obtain a non-natural chiral amino acid ester represented by formula (IV); S2) removing the protecting group from the non-natural chiral amino acid ester represented by formula (IV) to obtain a non-natural chiral amino acid containing cyanotriazoxide represented by formula (I) and a pharmaceutically acceptable salt thereof;

[0055]

[0056] Wherein, n is an integer of 0 to 5; R1 is an amino protecting group; R2 is a carboxyl protecting group; R is a substituted or unsubstituted C1 to C10 alkyl, cyano or nitro; X ​​is a heteroatom, a C1 to C10 alkylene; the substituent of the substituted C1 to C10 alkyl is one or more of halogen, cyano and nitro.

[0057] The present invention has no special restrictions on the sources of all raw materials, and they can be commercially available; the n, R and X are the same as described above and will not be repeated here; the R1 is preferably tert-butyloxycarbonyl; and R2 is preferably tert-butyl.

[0058] The compound represented by formula (II) is reacted with the compound represented by formula (III) in the presence of an acid-binding agent to obtain a non-natural chiral amino acid ester represented by formula (IV); the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is preferably 1: (0.8-1.2), more preferably 1: 1; the acid-binding agent is preferably an organic base, more preferably one or more of triethylamine (Et3N), N,N-diisopropylethylamine (DIPEA), N-methylmorpholine (NMM) and 4-dimethylaminopyridine (DMAP); the reaction is preferably carried out in an organic solvent; the organic solvent is an organic solvent well known to those skilled in the art without any special restrictions, and tetrahydrofuran is preferably used in the present invention; the compound represented by formula (II) is preferably first mixed with an organic solvent, and then the compound represented by formula (III) is added under low temperature conditions. The organic solution of the compound is prepared by heating the mixture to react; the temperature of the low temperature condition is preferably 0°C to 3°C; the reaction is preferably carried out at room temperature; the reaction time is preferably 4 to 8 hours, more preferably 4 to 6 hours; after the reaction, the solvent is preferably removed, and then ethyl acetate is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a desiccant, filtered, concentrated, and purified by chromatography to obtain a non-natural chiral amino acid ester represented by formula (IV); the number of washings with saturated brine is preferably 1 to 2 times; the desiccant is preferably anhydrous sodium sulfate; the chromatography separation and purification is preferably rapid silica gel column chromatography analysis; the elution solvent used for the chromatography separation is preferably petroleum ether and ethyl acetate; in the present invention, petroleum ether and ethyl acetate in a volume ratio of 10:1 are preferably used as the eluent, and then petroleum ether and ethyl acetate in a volume ratio of 2:1 are used as the eluent.

[0059] The non-natural chiral amino acid ester represented by formula (IV) is deprotected to obtain a non-natural chiral amino acid containing cyano triazoxide represented by formula (I) or a pharmaceutically acceptable salt thereof; the method for removing the protecting group is a method well known to those skilled in the art and is not particularly limited. In the present invention, the removal is preferably carried out under acidic conditions, specifically: the non-natural chiral amino acid ester represented by formula (IV) is mixed with an organic solvent, and then trifluoroacetic acid or concentrated hydrochloric acid is added for removal reaction to obtain a non-natural chiral amino acid containing cyano triazoxide represented by formula (I) or a pharmaceutically acceptable salt thereof; the organic solvent is an organic solvent well known to those skilled in the art and is not particularly limited. In the present invention, tetrahydrofuran is preferably used; the trifluoroacetic acid or concentrated hydrochloric acid is preferably added under low temperature conditions; the temperature of the low temperature conditions is preferably 0°C to 3°C; the molar ratio of the trifluoroacetic acid or concentrated hydrochloric acid to the compound represented by formula (II) is preferably (3 to 5): 1, more preferably 4: 1; the removal reaction is preferably carried out at room temperature; the removal reaction time is preferably 2 to 6 hours, more preferably 4 hours.

[0060] In a specific embodiment provided by the present invention, the non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof represented by formula (I) is reacted according to the following reaction formula:

[0061]

[0062] The present invention uses four cheap and readily available natural chiral L-α-amino acid derivatives as raw materials. The obtained novel non-natural chiral α-amino acid contains a cyanotriazine functional group, which undergoes a one-pot cycloaddition / oxidation reaction with a strained cyclooctenol compound under room temperature and air conditions to quickly generate the corresponding pyridine compound, and the secondary rate constant K2 reaches 975M -1 ·s -1 , providing a new class of unnatural chiral α-amino acids for bioorthogonal linking reactions, which can be used in chemical biology research.

[0063] The present invention also provides a non-natural chiral amino acid ester containing cyanotriazoxide, as shown in formula (IV):

[0064]

[0065] Wherein, n is an integer of 0 to 5; R1 is an amino protecting group; R2 is a carboxyl protecting group; R is a substituted or unsubstituted C1 to C10 alkyl, cyano or nitro; X ​​is a heteroatom, a C1 to C10 alkylene; the substituent of the substituted C1 to C10 alkyl is selected from one or more of halogen, cyano and nitro.

[0066] The n, R and X are the same as described above and will not be described again.

[0067] The present invention also provides an application of the non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, and the non-natural chiral amino acid ester containing cyanotriazoxide as a small molecule probe.

[0068] In a specific embodiment provided by the present invention, the non-natural chiral amino acid containing cyanotriazoxide or its pharmaceutically acceptable salt, the non-natural chiral amino acid ester containing cyanotriazoxide and the strained cyclooctenol compound can quickly undergo a cycloaddition reaction to generate pyridine compounds. Such non-natural chiral amino acids containing cyanotriazoxide and their derivatives can be used as small molecule probes for bioorthogonal linking reactions.

[0069] Furthermore, the above cycloaddition reaction is carried out at room temperature and in air.

[0070] To further illustrate the present invention, a non-natural chiral amino acid containing cyanotriazoxide, a preparation method thereof and an application thereof provided by the present invention are described in detail below in conjunction with examples.

[0071] The reagents used in the following examples are all commercially available and are used directly without further purification unless otherwise specified. All parts and percentages are by mass, and the temperatures are in degrees Celsius unless otherwise specified; the flash column chromatography uses Qingdao Ocean Chemical 200-300 mesh standard silica gel; the thin layer chromatography uses Qingdao Ocean Chemical 0.20 mm standard plate; the nuclear magnetic resonance spectroscopy data (NMR) is 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 single peak, d represents a double peak, t represents a triplet, q represents a quartet, and m represents a multiple peak).

[0072] Example 1: N 6 -[3-Cyano-6-(trifluoromethyl)-1,2,4-triazine-5-carbonyl]-l-lysine (Compound Ia)

[0073]

[0074] Add tert-butyloxycarbonyl-protected L-lysine tert-butyl ester (260 mg, 1.0 mmol) and tetrahydrofuran (5 mL) to the reaction flask, cool the reaction system to 0°C; add a solution of 3-cyano-6-trifluoromethyl-1,2,4-triazine-5-carbonyl chloride (237 mg, 1.0 mmol) in tetrahydrofuran (5 mL) to the reaction flask under stirring. After the addition, the mixture is warmed to room temperature and stirred for 6 hours. The reaction is complete when detected by thin layer chromatography. After the tetrahydrofuran solvent is removed by thin film rotary evaporation, the residue is dissolved in 30 mL of ethyl acetate and washed once with 15 mL of saturated brine. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and separated by rapid silica gel column chromatography (first using 10:1 volume ratio of petroleum ether and ethyl acetate as the elution solvent, and then using 2:1 volume ratio of petroleum ether and ethyl acetate as the elution solvent) to obtain the corresponding tert-butyloxycarbonyl-protected amino acid tert-butyl ester.

[0075] The tert-butyl ester of amino acid protected by tert-butyloxycarbonyl was dissolved in 15 mL of tetrahydrofuran, cooled to 0°C, and 37% concentrated hydrochloric acid (0.34 mL, 4.0 mmol) was added dropwise. After the addition was completed, the reaction system was warmed to room temperature for 4 hours. The reaction was complete after TLC. The solvent was removed by thin film rotary evaporation to obtain a light gray solid, which was compound Ia (364 mg, total yield 95%).

[0076] Compound Ia was analyzed by nuclear magnetic resonance to obtain 1H NMR (400MHz, DMSO-d6), δ (ppm): 1.30-1.35 (m, 2H), 1.57-1.65 (m, 2H), 1.70-1.76 (m, 2H), 3.34(t,2H),3.51(t,1H),8.75-8.81(br,2H),8.88-8.93(br,1H),12.11-12.33(br,1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm): –-62.0 (s, 3F).

[0077] Example 2: N 6 -(3,6-Dicyano-1,2,4-triazine-5-carboxylate)-l-lysine (Compound Ib)

[0078]

[0079] Add tert-butyloxycarbonyl-protected L-lysine tert-butyl ester (260 mg, 1.0 mmol) and tetrahydrofuran (5 mL) to the reaction flask, and cool the reaction system to 0°C; add a solution of 3,6-dicyano-1,2,4-triazine-5-carbonyl chloride (194 mg, 1.0 mmol) in tetrahydrofuran (5 mL) to the reaction flask under stirring. After the addition, the mixture is warmed to room temperature and stirred for 4 hours. The reaction is complete when detected by thin-layer chromatography. After the tetrahydrofuran solvent is removed by thin-film rotary evaporation, the residue is dissolved in 30 mL of ethyl acetate and washed once with 15 mL of saturated brine. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated, and separated by rapid silica gel column chromatography (first using 10:1 volume ratio of petroleum ether and ethyl acetate as the elution solvent, and then using 2:1 volume ratio of petroleum ether and ethyl acetate as the elution solvent) to obtain the corresponding tert-butyloxycarbonyl-protected amino acid tert-butyl ester.

[0080] The tert-butyl ester of amino acid protected by tert-butyloxycarbonyl was dissolved in 15 mL of tetrahydrofuran, cooled to 0°C, and trifluoroacetic acid (0.3 mL, 4.0 mmol) was added dropwise. After the addition was completed, the reaction system was warmed to room temperature for 4 hours. The reaction was complete when detected by thin layer chromatography. The solvent was removed by thin film rotary evaporation to obtain a light gray solid compound Ib (376 mg, total yield 90%).

[0081] Compound Ib was analyzed by nuclear magnetic resonance to obtain 1H NMR (400MHz, DMSO-d6), δ (ppm): 1.33-1.37 (m, 2H), 1.50-1.56 (m, 2H), 1.72-1.767 (m, 2H) ,3.30(t,2H),3.55(t,1H),8.70-8.79(br,2H),8.83-8.88(br,1H),12.01-12.30(br,1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm):-–73.97 (s, 3F).

[0082] Example 3: O-{2-[3-cyano-6-(trifluoromethyl)-1,2,4-triazine-5-carboxamido]ethylidene}-l-serine (Compound Ic)

[0083]

[0084] A similar method to Example 1 was used to prepare compound Ic (335 mg, total yield 87%).

[0085] Compound Ic was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6), δ (ppm): 3.30-3.41 (m, 4H), 3.63-3.71 (m, 3H), 8.90-8.95 (br, 3H), 12.30-12.36 (br, 1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm): –-61.7 (s, 3F).

[0086] Example 4: O-[2-(3,6-dicyano-1,2,4-triazine-5-carboxamido)ethylidene]-l-serine (Compound Id)

[0087]

[0088] A similar method to Example 1 was used to prepare compound Id (308 mg, total yield 90%).

[0089] The compound Id was analyzed by nuclear magnetic resonance and obtained 1 H NMR (400MHz, DMSO-d6), δ (ppm): 3.33-3.42 (m, 4H), 3.61-3.68 (m, 3H), 8.92-8.95 (br, 3H), 12.33-12.37 (br, 1H).

[0090] Example 5: S-{2-[3-cyano-6-(trifluoromethyl)-1,2,4-triazine-5-carboxamido]ethylidene}-l-cysteine ​​(Compound Ie)

[0091]

[0092] A similar method to Example 1 was used to prepare compound Ie (341 mg, total yield 85%).

[0093] Compound Ie was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6), δ (ppm): 2.89-3.03 (m, 4H), 3.31-3.38 (m, 2H), 3.70-3.74 (m, 1H), 8.91-8.94 (br, 3H), 12.11-12.19 (br, 1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm): –-62.4 (s, 3F).

[0094] Example 6: S-[2-(3,6-dicyano-1,2,4-triazine-5-carboxamido)ethylidene]-l-cysteine ​​(Compound If)

[0095]

[0096] A similar method to Example 1 was used to obtain compound If (315 mg, total yield 88%).

[0097] The compound If was analyzed by nuclear magnetic resonance, and the 1 H NMR (400MHz, DMSO-d6), δ (ppm): 2.90-3.05 (m, 4H), 3.35-3.40 (m, 2H), 3.71-3.74 (m, 1H), 8.90-8.94 (br, 3H), 12.18-12.27 (br, 1H).

[0098] Example 7: Se-{2-[3-cyano-6-(trifluoromethyl)-1,2,4-triazine-5-carboxamido]ethylidene}-l-cysteine ​​(Compound Ig)

[0099]

[0100] A similar method to Example 1 was used to obtain compound Ig (398 mg, total yield 89%).

[0101] The compound Ig was analyzed by nuclear magnetic resonance and obtained 1H NMR (400MHz, DMSO-d6), δ (ppm): 1.77-1.90 (m, 4H), 3.01-3.10 (m, 2H), 3.52-3.58 (m, 1H), 8.76-8.85 (br, 3H), 12.20-12.26 (br, 1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm):-–61.9 (s, 3F).

[0102] Example 8: Se-[2-(3,6-dicyano-1,2,4-triazine-5-carboxamido)ethylidene]-l-cysteine ​​(Compound Ih)

[0103]

[0104] A similar method to Example 1 was used to prepare compound Ih (372 mg, total yield 92%).

[0105] Compound Ih was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6), δ (ppm): 1.79-1.90 (m, 4H), 3.05-3.12 (m, 2H), 3.55-3.59 (m, 1H), 8.71-8.78 (br, 3H), 12.21-12.26 (br, 1H).

[0106] Example 9: N 5 -[3-cyano-6-(trifluoromethyl)-1,2,4-triazine-5-carbonyl]-l-ornithine (Compound Ii)

[0107]

[0108] A similar method to Example 2 was used to obtain compound Ii (406 mg, total yield 91%).

[0109] Compound Ii was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6), δ (ppm): 1.67-1.74 (m, 4H), 2.94-3.02 (m, 2H), 3.41-3.45 (m, 1H), 8.88-8.91 (br, 3H), 12.12-12.17 (br, 1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm): –62.0 (s, 3F); –73.95 (s, 3F).

[0110] Example 10: N 5-(3,6-dicyano-1,2,4-triazine-5-carboxylate)-l-ornithine (Compound Ij)

[0111]

[0112] A similar method to Example 2 was used to obtain compound Ij (47 mg, total yield 86%).

[0113] Compound Ij was analyzed by nuclear magnetic resonance to obtain 1 H NMR (400MHz, DMSO-d6), δ (ppm): 1.70-1.77 (m, 4H), 2.98-3.08 (m, 2H), 3.44-3.49 (m, 1H), 8.90-8.95 (br, 3H), 12.17-12.22 (br, 1H); 19 F NMR (376MHz, DMSO-d6), δ (ppm): –73.90 (s, 3F).

[0114] Bioorthogonal ligation chemistry assay

[0115] The one-pot cycloaddition / oxidation reaction of unnatural chiral amino acid tert-butyl esters containing cyanotriazoxide and strained cyclooctenols (TCO and s-TCO) was carried out, and the secondary kinetic rate constant was determined by ultraviolet (UV)-stopped-flow spectrometer. The unnatural chiral amino acid tert-butyl ester containing cyanotriazoxide was prepared in acetonitrile / water (1 / 1) at four concentrations: 0.1mM, 0.075mM, 0.05mM, and 0.025mM. In the experiment, the molar ratio of strained cyclooctenol compounds to unnatural chiral amino acid tert-butyl ester containing cyanotriazoxide 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.

[0116] Table 1 Secondary kinetic rate constant test results

[0117]

[0118]

[0119] The data in the above table show that non-natural chiral amino acid tert-butyl ester containing cyanotriazoxide can rapidly undergo an inverse electron demand Diels-Alder cycloaddition (IEDDA) reaction with strained cyclooctenol compounds, proving that such compounds can be used in bioorthogonal ligation chemistry experiments and have great potential application value in chemical biology and bioimaging.

[0120] 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 non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, characterized in that: Including the structure shown in formula (I): Wherein, n is an integer from 0 to 5; R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro; X is selected from heteroatoms, C1-C10 alkylene groups; The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

2. The non-natural chiral amino acid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The n is 0 or 1.

3. The unnatural chiral amino acid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R is selected from substituted or unsubstituted C1-C5 alkyl, cyano or nitro; the substituent of the substituted C1-C5 alkyl is selected from one or more of halogen, cyano and nitro.

4. The unnatural chiral amino acid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R is selected from trifluoromethyl, cyano or nitro; And / or, X is selected from oxygen, sulfur, selenium, and C1-C5 alkylene.

5. The unnatural chiral amino acid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Including one of the structures shown in Ia~Ij:

6. The unnatural chiral amino acid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pharmaceutically acceptable salt is trifluoroacetate or hydrochloride.

7. A method for preparing a non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof, characterized in that: The following steps are involved: S1) reacting the compound represented by formula (II) with the compound represented by formula (III) in the presence of an acid-binding agent to obtain a non-natural chiral amino acid ester represented by formula (IV); S2) removing the protecting group from the non-natural chiral amino acid ester represented by formula (IV) to obtain the non-natural chiral amino acid containing cyanotriazoxide represented by formula (I) or a pharmaceutically acceptable salt thereof; Wherein, n is an integer from 0 to 5; R1 is an amino protecting group; R2 is a carboxyl protecting group; R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro; X is selected from heteroatoms, C1-C10 alkylene groups; The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

8. The preparation method according to claim 7, characterized in that: R1 is tert-butyloxycarbonyl; R2 is tert-butyl; The acid binding agent is selected from one or more of triethylamine, N,N-diisopropylethylamine, N-methylmorpholine and 4-dimethylaminopyridine.

9. A non-natural chiral amino acid ester containing cyanotriazoxide, characterized in that: As shown in formula (IV): Wherein, n is an integer from 0 to 5; R1 is an amino protecting group; R2 is a carboxyl protecting group; R is selected from substituted or unsubstituted C1-C10 alkyl, cyano or nitro; X is selected from heteroatoms, C1-C10 alkylene groups; The substituent of the substituted C1-C10 alkyl group is selected from one or more of halogen, cyano and nitro.

10. Use of the non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 6, the non-natural chiral amino acid containing cyanotriazoxide or a pharmaceutically acceptable salt thereof prepared by the preparation method of any one of claims 7 to 8, or the non-natural chiral amino acid ester containing cyanotriazoxide as described in claim 9 as a small molecule probe.

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