Unnatural chiral amino acids containing cyanotriazenes, methods of making and using same

Abstract

The application provides a non-natural chiral amino acid containing a cyano-triazine 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 a cyano-triazine or the pharmaceutically acceptable salt thereof provided by the application can be subjected to a one-pot cycloaddition / oxidation reaction with a strain cyclooctene alcohol compound under room temperature air conditions, and a corresponding pyridine compound is quickly generated, and the secondary rate constant K2 can reach 975 M ‑1 ·s ‑1 A new type of non-natural chiral alpha-amino acid is provided for a biological orthogonal linking reaction, and can be further applied to chemical biology research based on the biological orthogonal linking reaction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of unnatural chiral amino acids, and particularly relates to an unnatural chiral amino acid containing a cyanotriazene, a preparation method and application thereof. BACKGROUND

[0002] Amino acids are the basic substances for life, and their synthesis and application have also attracted extensive attention of scientific researchers. Proteins in living organisms are composed of a limited number of 20 natural chiral α-amino acids, which greatly limits the application range of protein chemistry and function. In recent years, the gene code expansion (GCE) technology has realized the introduction of unnatural amino acids into specific sites of any target protein in a living organism. This technology uses an aminoacyl-tRNA synthetase / tRNA pair (aaRS / tRNA pairs) that is orthogonal to the host cell organism, and encodes unnatural amino acids through a stop codon (UAG / UGA / UAA) (Peter G. Schultz et al., Annu. Rev. Biochem. 2010, 79, 413-44). So far, more than 200 unnatural chiral α-amino acids have been introduced into polypeptides or proteins by the gene code expansion technology; and all the unnatural chiral α-amino acids with a biological orthogonal functional group are derived from natural aromatic amino acids (such as phenylalanine, tryptophan, tyrosine and histidine), serine, threonine, (semi)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 attempted to use the inverse electron-demand Diels-Alder cycloaddition (IED-DA) of 1,2,4-triazines with strained cyclooctyne alcohols to develop new bioorthogonal reactions (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; & J.-A. Ma, et al., ACS Catal. 2019, 9, 4600-4608). However, in these inverse electron-demand Diels-Alder cycloadditions of 1,2,4-triazines with strained cyclooctyne alcohols, the highest second-order rate constant K2 is only 100 M -1 ·s -1 Therefore, there is an urgent need to design and synthesize unnatural chiral amino acids containing novel triazine heterocycles to improve their reaction efficiency. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a cyanotriazine-containing unnatural chiral amino acid, a preparation method and application thereof, which has a high cycloaddition reaction rate with strained cyclooctyne alcohols at room temperature.

[0005] The present application provides a cyanotriazine-containing unnatural chiral amino acid or a pharmaceutically acceptable salt thereof, comprising a structure represented by 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 a heteroatom and C1-C10 alkylene;

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

[0011] Preferably, n is 0 or 1.

[0012] Preferably, 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, R is selected from trifluoromethyl, cyano or nitro.

[0014] Preferably, X is selected from oxygen, sulfur, selenium, C1-C5 alkylene.

[0015] Preferably, one of the structures represented by I-a to I-j is included:

[0016]

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

[0018] The present application also provides a preparation method of a non-natural chiral amino acid containing a cyano-triazene or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0019] 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);

[0020] S2) removing the protecting group of the non-natural chiral amino acid ester represented by formula (IV) to obtain a non-natural chiral amino acid containing a cyano-triazene 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 a heteroatom, C1-C10 alkylene;

[0026] The substituent of the substituted C1-C10 alkyl 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 application also provides a non-natural chiral amino acid ester containing a cyano-triazine, as shown in formula (IV):

[0030]

[0031] 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 a heteroatom, C1-C10 alkylene;

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

[0036] The application also provides a use of the above-mentioned non-natural chiral amino acid containing a cyano-triazine or a pharmaceutically acceptable salt thereof or the above-mentioned non-natural chiral amino acid ester containing a cyano-triazine as a small molecule probe.

[0037] The application provides a non-natural chiral amino acid containing a cyano-triazine or a pharmaceutically acceptable salt thereof, comprising a structure shown in formula (I). Compared with the prior art, the non-natural chiral amino acid containing a cyano-triazine or a pharmaceutically acceptable salt thereof provided by the application can perform one-pot cycloaddition / oxidation reaction with a tensioned cyclooctene alcohol compound at room temperature under air condition, and quickly generate a corresponding pyridine compound, and the secondary rate constant K2 can reach 975 M -1 ·s -1 A new type of non-natural chiral alpha-amino acid is provided for biological orthogonal ligation reaction, and can be further applied to chemical biology research based on biological orthogonal ligation reaction. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

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

[0040]

[0041] 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 a heteroatom, C1-C10 alkylene;

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

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

[0046] In one specific embodiment provided by the present application, R is preferably 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; and the halogen is any halogen known to those skilled in the art without any particular limitation, and is preferably fluorine, chlorine or bromine in the present application.

[0047] In one specific embodiment provided by the present application, R is preferably 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; and the halogen is any halogen known to those skilled in the art without any particular limitation, and is preferably fluorine, chlorine or bromine in the present application.

[0048] In one specific embodiment provided by the present application, R is preferably 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; and the halogen is any halogen known to those skilled in the art without any particular limitation, and is preferably fluorine, chlorine or bromine in the present application.

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

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

[0051] In one specific embodiment provided by the present application, the unnatural chiral amino acid or the pharmaceutically acceptable salt thereof comprises one of the structures represented by I-a to I-j:

[0052]

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

[0054] The present application also provides a preparation method of the unnatural chiral amino acid containing cyanotriazene or pharmaceutically acceptable salt thereof, comprising the following steps: S1) reacting a compound shown in formula (II) with a compound shown in formula (III) in the presence of an acid binding agent to obtain an unnatural chiral amino acid ester shown in formula (IV); S2) removing the protecting group of the unnatural chiral amino acid ester shown in formula (IV) to obtain the unnatural chiral amino acid containing cyanotriazene shown in formula (I) and pharmaceutically acceptable salt thereof.

[0055]

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

[0057] The present application does not have special restrictions on the source of all raw materials, which can be commercially available; n, R and X are the same as described above, which will not be described here; R1 is preferably tert-butyloxycarbonyl; R2 is preferably tert-butyl.

[0058] The compound shown as formula (II) is reacted with the compound shown as formula (III) in the presence of an acid binding agent to obtain the non-natural chiral amino acid ester shown as formula (IV); the molar ratio of the compound shown as formula (II) to the compound shown as 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 can be any organic solvent known to those skilled in the art without special limitation, and in the present application, tetrahydrofuran is preferred; the compound shown as formula (II) is preferably first mixed with an organic solvent, then an organic solution of the compound shown as formula (III) is added under low temperature conditions, and the reaction is carried out after warming; the temperature of the low temperature conditions is preferably 0-3°C; the reaction is preferably carried out at room temperature; the reaction time is preferably 4-8 h, more preferably 4-6 h; after the reaction is completed, the solvent is preferably removed, then ethyl acetate is added to obtain an organic phase; the organic phase is washed with saturated brine, dried with a drying agent, filtered, concentrated, purified by chromatography to obtain the non-natural chiral amino acid ester shown as formula (IV); the number of times of washing with saturated brine is preferably 1-2 times; the drying agent is preferably anhydrous sodium sulfate; the chromatography purification is preferably flash silica gel column chromatography; the elution solvent used in the chromatography purification is preferably petroleum ether and ethyl acetate; in the present application, petroleum ether and ethyl acetate in a volume ratio of 10:1 are used as the eluent, then petroleum ether and ethyl acetate in a volume ratio of 2:1 are used as the eluent.

[0059] The protecting group of the non-natural chiral amino acid ester shown as formula (IV) is removed to obtain the non-natural chiral amino acid containing a cyanotriazine shown as formula (I) or a pharmaceutically acceptable salt thereof; the method for removing the protecting group can be any method known to those skilled in the art without special limitation, and in the present application, the removal is preferably carried out under acidic conditions, specifically: the non-natural chiral amino acid ester shown as formula (IV) is mixed with an organic solvent, then trifluoroacetic acid or concentrated hydrochloric acid is added to carry out the removal reaction to obtain the non-natural chiral amino acid containing a cyanotriazine shown as formula (I) or a pharmaceutically acceptable salt thereof; the organic solvent can be any organic solvent known to those skilled in the art without special limitation, and in the present application, tetrahydrofuran is preferred; the trifluoroacetic acid or concentrated hydrochloric acid is preferably added under low temperature conditions; the temperature of the low temperature conditions is preferably 0-3°C; the molar ratio of the trifluoroacetic acid or concentrated hydrochloric acid to the compound shown as formula (II) is preferably (3-5):1, more preferably 4:1; the removal reaction is preferably carried out at room temperature; the removal reaction time is preferably 2-6 h, more preferably 4 h.

[0060] In one specific embodiment provided by the present application, the non-natural chiral amino acid containing cyanotriazine of formula (I) or its pharmaceutically acceptable salt is prepared according to the following reaction formula:

[0061]

[0062] The present application uses four cheap and readily available natural chiral L-alpha-amino acid derivatives as raw materials to obtain a new non-natural chiral alpha-amino acid containing cyanotriazine functional group, which can quickly generate the corresponding pyridine compound by one-pot cycloaddition / oxidation reaction with a tension ring octene alcohol compound at room temperature under air conditions, and the secondary rate constant K2 reaches 975M -1 ·s -1 , which provides a new type of non-natural chiral alpha-amino acid for biological orthogonal linking reaction, and can be used for chemical biology research.

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

[0064]

[0065] , wherein n is an integer from 0 to 5; R1 is an amino protecting group; R2 is a carboxyl protecting group; R is a substituted or unsubstituted C1-C10 alkyl, cyano or nitro; X is a heteroatom, C1-C10 alkylene; the substituent of the substituted C1-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 here.

[0067] The present application also provides the use of the above-mentioned non-natural chiral amino acid containing cyanotriazine or its pharmaceutically acceptable salt, non-natural chiral amino acid ester containing cyanotriazine as a small molecule probe.

[0068] In one specific embodiment provided by the present application, the non-natural chiral amino acid containing cyanotriazine or its pharmaceutically acceptable salt, non-natural chiral amino acid ester containing cyanotriazine can quickly perform cycloaddition reaction with tension ring octene alcohol compound to generate pyridine compound, and such non-natural chiral amino acid containing cyanotriazine and its derivatives as a small molecule probe can be used for biological orthogonal linking reaction.

[0069] Further, the above-mentioned cycloaddition reaction is carried out at room temperature and under air conditions.

[0070] In order to further illustrate the present application, the following embodiments are used to describe in detail the non-natural chiral amino acid containing cyanotriazine, its preparation method and application provided by the present application.

[0071] All reagents used in the following examples were commercially available and used directly without further purification, unless otherwise noted. All parts and percentages are by mass parts and mass percentages, and temperatures are in degrees Celsius, unless otherwise noted; flash column chromatography was performed using 200-300 mesh standard silica gel from Qingdao Haizhuan Chemicals; thin layer chromatography was performed using 0.20 mm standard plates from Qingdao Haizhuan Chemicals; nuclear magnetic resonance spectroscopy data (NMR) were obtained using a Bruker 400 MHz spectrometer, with tetramethylsilane as the internal standard and deuterated chloroform as the solvent (s indicates singlet, d indicates doublet, t indicates triplet, q indicates quartet, and m indicates multiplet).

[0072] Example 1: N 6 -[3-cyano-6-(trifluoromethyl)-1,2,4-triazine-5-formyl]-l-lysine (Compound I-a)

[0073]

[0074] tert-Butyl tert-butyl ester of L-lysine (260 mg, 1.0 mmol) and tetrahydrofuran (5 mL) were added to a reaction flask, and the reaction system was cooled to 0 °C; a solution of 3-cyano-6-trifluoromethyl-1,2,4-triazine-5-formyl chloride (237 mg, 1.0 mmol) in tetrahydrofuran (5 mL) was added dropwise to the reaction flask with stirring. After the addition was complete, the mixture was allowed to warm to room temperature and stirring was continued for 6 hours, after which thin layer chromatography indicated that the reaction was complete. The tetrahydrofuran solvent was removed by thin film rotary evaporation, and the residue was dissolved in 30 mL of ethyl acetate and washed once with 15 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by flash column chromatography on silica gel (first using 10:1 by volume of petroleum ether and ethyl acetate as the eluent, and then using 2:1 by volume of petroleum ether and ethyl acetate as the eluent) to obtain the corresponding tert-butyloxy carbonyl-protected amino acid tert-butyl ester.

[0075] The tert-butyloxy carbonyl-protected amino acid tert-butyl ester was dissolved in 15 mL of tetrahydrofuran, cooled to 0 °C, and then 37% concentrated hydrochloric acid (0.34 mL, 4.0 mmol) was added dropwise. After the addition was complete, the reaction system was allowed to warm to room temperature and react for 4 hours, after which thin layer chromatography indicated that the reaction was complete. The solvent was removed by thin film rotary evaporation to obtain a light gray solid, which was Compound I-a (364 mg, total yield 95%).

[0076] Compound I-a was analyzed using a nuclear magnetic resonance spectrometer to obtain 1H NMR (400 MHz, DMSO-d6), d (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 (376 MHz, DMSO-d6), d (ppm): -62.0 (s, 3F).

[0077] Example 2: N 6 -(3,6-dicyano-1,2,4-triazine-5-formyl)-l-lysine (Compound I-b)

[0078]

[0079] tert-Butyl tert-butyl (tert-butoxycarbonyl) protected L-lysinate (260 mg, 1.0 mmol) and tetrahydrofuran (5 mL) were added to a reaction flask, and the reaction system was cooled to 0 °C; a solution of 3,6-dicyano-1,2,4-triazine-5-formyl chloride (194 mg, 1.0 mmol) in tetrahydrofuran (5 mL) was added dropwise to the reaction flask with stirring. After the addition was completed, the mixture was allowed to warm to room temperature and the reaction was continued for 4 hours with stirring. Thin layer chromatography showed that the reaction was complete. After removing the tetrahydrofuran solvent by thin film rotary evaporation, the residue was dissolved in 30 mL of ethyl acetate and washed once with 15 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by flash silica gel column chromatography (firstly using 10:1 volume ratio of petroleum ether and ethyl acetate as eluent, and then using 2:1 volume ratio of petroleum ether and ethyl acetate as eluent) to obtain the corresponding tert-butyloxy carbonyl-protected amino acid tert-butyl ester.

[0080] The tert-butyloxy carbonyl-protected amino acid tert-butyl ester was dissolved in 15 mL of tetrahydrofuran, cooled to 0 °C, and then trifluoroacetic acid (0.3 mL, 4.0 mmol) was added dropwise. After the addition was completed, the reaction system was allowed to warm to room temperature and the reaction was continued for 4 hours. Thin layer chromatography showed that the reaction was complete. The solvent was removed by thin film rotary evaporation to obtain light gray solid compound I-b (376 mg, total yield 90%).

[0081] Compound I-b was analyzed by nuclear magnetic resonance instrument to obtain 1H NMR (400 MHz, 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 (376 MHz, DMSO-d6), δ (ppm): - -73.97 (s, 3F).

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

[0083]

[0084] In a similar manner to Example 1, Compound I-c was prepared (335 mg, overall yield 87%).

[0085] Compound I-c was analyzed using a nuclear magnetic resonance instrument to obtain 1 H NMR (400 MHz, 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). 19 F NMR (376 MHz, DMSO-d6), δ (ppm): - -61.7 (s, 3F).

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

[0087]

[0088] In a similar manner to Example 1, Compound I-d was prepared (308 mg, overall yield 90%).

[0089] Compound I-d was analyzed using a nuclear magnetic resonance instrument to obtain 1 H NMR (400 MHz, 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-triazin-5- carboxamido]ethylidene}-l-cysteine (Compound I-e)

[0091]

[0092] Compound I-e was prepared in a similar manner as in Example 1 (341 mg, 85% overall yield).

[0093] Compound I-e was analyzed by NMR to give 1 H NMR (400 MHz, 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 (376 MHz, DMSO-d6), δ (ppm): -62.4 (s, 3F).

[0094] Example 6: S-[2-(3,6-dicyano-1,2,4-triazin-5-carboxamido)ethylidene]-l- cysteine (Compound I-f)

[0095]

[0096] Compound I-f was prepared in a similar manner as in Example 1 (315 mg, 88% overall yield).

[0097] Compound I-f was analyzed by NMR to give 1 H NMR (400 MHz, 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-triazin-5- carboxamido]ethylidene}-l-cysteine (Compound I-g)

[0099]

[0100] Compound I-g was prepared in a similar manner as in Example 1 (398 mg, 89% overall yield).

[0101] Compound I-g was analyzed by NMR to give 1H NMR (400 MHz, DMSO-d6), d (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 (376 MHz, DMSO-d6), d (ppm): -61.9 (s, 3F).

[0102] Example 8: Se-[2-(3,6-dicyano-l,2,4-triazin-5-carboxamido)ethyl]-l-cysteine (Compound I-h)

[0103]

[0104] In a similar manner to Example 1, Compound I-h was prepared (372 mg, 92% overall yield).

[0105] Compound I-h was analyzed using a nuclear magnetic resonance instrument to obtain 1 H NMR (400 MHz, DMSO-d6), d (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);

[0106] Example 9: N 5 -[3-cyano-6-(trifluoromethyl)-l,2,4-triazin-5-carbonyl]-l-ornithine (Compound I-i)

[0107]

[0108] In a similar manner to Example 2, Compound I-i was prepared (406 mg, 91% overall yield).

[0109] Compound I-i was analyzed using a nuclear magnetic resonance instrument to obtain 1 H NMR (400 MHz, DMSO-d6), d (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 (376 MHz, DMSO-d6), d (ppm): -61.9 (s, 3F).

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

[0111]

[0112] In a similar manner to Example 2, Compound I-j was prepared (47 mg, 86% overall yield).

[0113] Compound I-j was analyzed by nuclear magnetic resonance instrument to obtain 1 H NMR (400 MHz, 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 (376 MHz, DMSO-d6), δ (ppm): -73.90 (s, 3F).

[0114] Bioorthogonal ligation chemistry test

[0115] The one-pot cycloaddition / oxidation reaction of cyanotriazene-containing unnatural chiral amino acid tert-butyl ester and TCO and s-TCO was carried out, and the second-order kinetic rate constant was determined by UV-stopped-flow device. The cyanotriazene-containing unnatural chiral amino acid tert-butyl ester was prepared in acetonitrile / water (1 / 1) at four concentrations: 0.1 mM, 0.075 mM, 0.05 mM, 0.025 mM, and the molar ratio of the unnatural chiral amino acid of the cyanotriazene-containing unnatural chiral amino acid tert-butyl ester to the TCO and s-TCO compound was 20 / 1 in the experiment, each group of experiments was determined for 3 times to take the average value, and the second-order kinetic rate constant K2(M -1 ·s -1 ) is shown in Table 1.

[0116] Table 1: Second-order kinetic rate constant test results

[0117]

[0118]

[0119] The data in the above table shows that the cyanotriazene-containing unnatural chiral amino acid tert-butyl ester and the TCO and s-TCO compound can quickly undergo inverse electron demand Diels-Alder cycloaddition reaction (IEDDA), which proves that such compounds can be used for bioorthogonal ligation chemistry test, and have great potential application value in chemical biology and biological imaging.

[0120] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A non-natural chiral amino acid containing cyanotriazine or a pharmaceutically acceptable salt thereof, characterized in that, Including the structure shown in equation (I): Where n is an integer from 0 to 5; R is selected from substituted or unsubstituted C1~C3 alkyl or cyano groups; X is selected from oxygen, sulfur, selenium, and C1-C3 alkylene groups; The substituents of the substituted C1-C3 alkyl group are selected from halogens or cyano groups; the halogen is fluorine.

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

3. The non-natural chiral amino acid or its pharmaceutically acceptable salt according to claim 1, characterized in that, The R is selected from trifluoromethyl, cyano, or nitro; And / or, the X is selected from oxygen, sulfur, selenium, and C1-C5 alkylene groups.

4. The non-natural chiral amino acid or its pharmaceutically acceptable salt according to claim 1, characterized in that, Including one of the structures shown in Ia~Ij: 。 5. The non-natural chiral amino acid or its pharmaceutically acceptable salt according to claim 1, characterized in that, The pharmaceutically acceptable salt is trifluoroacetate or hydrochloride.

6. A method for preparing a non-natural chiral amino acid containing cyanotriazine or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: S1) The compound shown in formula (II) is reacted with the compound shown in formula (III) in the presence of an acid-binding agent to obtain the non-natural chiral amino acid ester shown in formula (IV); S2) Remove the protecting group from the non-natural chiral amino acid ester of formula (IV) to obtain the non-natural chiral amino acid containing cyanotriazine or a pharmaceutically acceptable salt thereof, as shown in formula (I); Where 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~C3 alkyl or cyano groups; X is selected from oxygen, sulfur, selenium, and C1-C3 alkylene groups; The substituents of the substituted C1-C3 alkyl group are selected from halogens or cyano groups; the halogen is fluorine.

7. The preparation method according to claim 6, 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.

8. A non-natural chiral amino acid ester containing cyanotriazine, characterized in that, As shown in equation (IV): Where 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~C3 alkyl or cyano groups; X is selected from oxygen, sulfur, selenium, and C1-C3 alkylene groups; The substituents of the substituted C1-C3 alkyl group are selected from halogens or cyano groups; the halogen is fluorine.

9. The application of the non-natural chiral amino acid containing cyanotriazine or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, the non-natural chiral amino acid containing cyanotriazine or a pharmaceutically acceptable salt thereof prepared by any one of claims 6 to 7, or the non-natural chiral amino acid ester containing cyanotriazine as described in claim 8, in the preparation of small molecule probes for bioorthogonal linkage reactions; wherein the bioorthogonal linkage reaction is a cycloaddition reaction of the non-natural chiral amino acid containing cyanotriazine or a pharmaceutically acceptable salt thereof, the non-natural chiral amino acid ester containing cyanotriazine, and a strained octenol compound to generate a pyridine compound.

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