A compound, a method of preparation and use for preparing derivatizing reagents

Abstract

The application discloses a compound, a preparation method and a use for preparing a derivatization reagent, and belongs to the field of chemistry. The application claims a compound TMT-mAla-PP with a novel chemical structure and a preparation method and a use thereof. Compared with the same compound TMT-PP or TMT-Pro-PP in the prior art, the derivatization product obtained by using the TMT-mAla-PP provided by the application as a derivatization reagent for performing a derivatization reaction on a bile acid compound has a significantly better mass spectrometry response sensitivity. Therefore, the compound provided by the application can be used for preparing a derivatization reagent for a bile acid compound, and the derivatization reagent can perform derivatization modification on the bile acid compound before mass spectrometry detection of the bile acid compound, so that the detection performance of the mass spectrometry on the bile acid compound is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of chemistry and relates to the synthesis and application of novel structural compounds, specifically to a compound, its preparation method, and its use in preparing derivatization reagents. Background Technology

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) combines the superior separation performance of liquid chromatography with the high selectivity and sensitivity of mass spectrometry, making it the mainstream technology platform for the analysis of trace small molecule compounds in complex matrices. However, this technology still has significant limitations in the direct detection of small molecules: some target compounds lack ionizable functional groups, resulting in low ionization efficiency; compounds with rigid structures are difficult to generate characteristic and stable daughter ions during collision-induced dissociation; and compounds with poor chemical structural stability are prone to degradation within the ion source. These problems severely affect detection sensitivity and the reliability of qualitative and quantitative analysis, significantly increasing the difficulty of accurate quantitative analysis.

[0003] Derivatization reagents can be used to structurally modify target analytes, significantly improving the detection sensitivity of liquid chromatography-tandem mass spectrometry (LC-MS / MS), optimizing chromatographic separation behavior, and effectively enhancing quantitative accuracy in the absence of stable isotopic internal standards. This is an effective strategy to overcome the aforementioned technical bottlenecks. Utilizing the high selectivity of derivatization reagents for specific functional groups and the characteristic fragmentation patterns of derivatized products, matrix interference can be significantly reduced and the selectivity of the detection method enhanced. Introducing hydrophobic or hydrophilic groups allows for flexible control of the retention behavior of target analytes on C18 columns, while introducing easily ionized groups can significantly enhance mass spectrometry ionization efficiency and response intensity, thus meeting the high-sensitivity analysis requirements of trace metabolites in complex matrices. The selection of derivatization reagents mainly depends on the reactive functional groups contained in the analyte and the research objectives. Ideal derivatization reagents should possess core characteristics such as high reactivity, excellent selectivity, good availability, and outstanding detection sensitivity.

[0004] Carboxylic acids, as key small-molecule metabolites in vivo, are a focus of research in liquid chromatography-tandem mass spectrometry (LC-MS / MS) derivatization analysis. Bile acids belong to a class of highly homologous C24 steroidal carboxylic acids, encompassing free bile acids and various conjugates formed with glycine, taurine, etc. Abnormal levels of bile acids in vivo are closely related to various hepatobiliary diseases and metabolic disorders, possessing important biological functions and clinical diagnostic value. Although free bile acids readily form deprotonated molecular ions in negative ion detection mode, they struggle to generate stable and characteristic fragment ions during collision-induced dissociation. This results in significant shortcomings in detection sensitivity, selectivity, and limit of quantitation, hindering the highly sensitive and accurate analysis of low-abundance bile acids. Furthermore, the sensitizing effect of existing derivatization reagents on the mass spectrometry signal of bile acids still needs further improvement.

[0005] In order to overcome the shortcomings of the existing technology, this invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound, a preparation method, and its use in preparing derivatization reagents.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A compound with the following chemical structural formula:

[0009] .

[0010] A method for preparing the above-mentioned compound, comprising the following synthetic route:

[0011] .

[0012] In one specific embodiment, the preparation method of intermediate T1 includes: adding 6-chloro-2,4-diamino-1,3,5-triazine to a reaction vessel, adding sodium hydride and DMF, adding iodomethane while stirring, and stirring the reaction at room temperature; after the reaction is completed, transferring the reaction solution to a separatory funnel, adding water and extracting with ethyl acetate, collecting the ethyl acetate phase, removing water, concentrating, and purifying by normal-phase silica gel column chromatography to obtain intermediate T1.

[0013] In one specific embodiment, the preparation method of intermediate T2 includes: adding L-alanine and sodium carbonate to a reaction vessel and dissolving them in water; adding intermediate T1 under stirring and heating conditions; heating and refluxing reaction; stopping heating after the reaction is completed; transferring the reaction vessel to an ice bath for cooling; back-extracting with dichloromethane; retaining the aqueous phase; adjusting the pH to 3-4; extracting with dichloromethane; collecting the organic phase; removing water; concentrating to obtain intermediate T2.

[0014] In one specific embodiment, the preparation method of intermediate T3 includes: adding intermediate T2 to a reaction vessel, dissolving it in anhydrous acetonitrile, and then sequentially adding N-methylimidazole, N-Boc-piperazine, and TCFH, stirring the reaction at room temperature; after the reaction is completed, evaporating the reaction solution to obtain a crude product, and purifying it by normal-phase silica gel column chromatography to obtain intermediate T3.

[0015] Preferably, the preparation method of intermediate T4 includes: adding intermediate T3, sodium hydride, and DMF to a reaction vessel, adding iodomethane dropwise while stirring, and stirring the reaction at room temperature; after the reaction is completed, adding water to the reaction solution and extracting with ethyl acetate, collecting the organic phase, removing water, concentrating, obtaining the crude product, and purifying the crude product by normal phase silica gel column chromatography to obtain intermediate T4.

[0016] In one specific embodiment, the preparation method of the target compound TMT-mAla-PP includes: adding intermediate T4 to a reaction vessel, adding dichloromethane to dissolve it, adding trifluoroacetic acid while stirring, and stirring the reaction at room temperature; after the reaction is completed, the reaction solution is first washed with saturated NaHCO3 solution, then washed with water to remove water from the organic phase, and purified by normal phase silica gel column chromatography to obtain the final product.

[0017] The above compounds are used in the preparation of derivatization reagents.

[0018] In one specific embodiment, the derivatizing reagent is used to derivatize the bile acid compound before mass spectrometry detection of the bile acid compound.

[0019] Beneficial effects:

[0020] This invention provides a novel chemical compound, TMT-mAla-PP, and its preparation method. Compared with similar compounds TMT-PP or TMT-Pro-PP in the prior art, the derivatized products obtained by using TMT-mAla-PP as a derivatizing reagent to derivatize bile acid compounds exhibit significantly superior mass spectrometry response sensitivity. Therefore, the compound provided by this invention can be used to prepare derivatizing reagents for bile acid compounds. These reagents can be used to derivatize and modify bile acid compounds before mass spectrometry detection, thereby significantly improving the detection performance of mass spectrometry for bile acid compounds. Attached Figure Description

[0021] Figure 1 This is the mass spectrometry detection chromatogram of compound TMT-mAla-PP in Example 1;

[0022] Figure 2 The 1H NMR spectrum of compound TMT-mAla-PP in Example 1;

[0023] Figure 3 A comparison of the chemical structures of TMT-mAla-PP, TMT-PP, and TMT-Pro-PP;

[0024] Figure 4 The mass spectrometry peaks of the derivatives obtained by derivatization of six bile acid compounds with TMT-PP as the derivatizing reagent are shown.

[0025] Figure 5 The mass spectrometry response peaks of the derivatives obtained by derivatization of six bile acid compounds with TMT-Pro-PP as the derivatization reagent are shown.

[0026] Figure 6The mass spectrometry peaks of the derivatives obtained by derivatizing six bile acid compounds with TMT-mAla-PP as the derivatizing reagent are shown. Detailed Implementation

[0027] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0028] Example 1: Synthesis and structural confirmation of compound TMT-mAla-PP

[0029] Synthesis route:

[0030]

[0031] Synthesis steps:

[0032] 1. Preparation of intermediate 6-chloro-N2,N2,N4,N4-tetramethyl-1,3,5-triazine-2,4-diamine (T1)

[0033] Add 400 mg (2.748 mmol) of 6-chloro-2,4-diamino-1,3,5-triazine and 400 mg (16.667 mmol) of sodium hydride to a 100 mL round-bottom flask. Add 20 mL of DMF and stir with a magnetic stirrer. Add 1.5 mL of iodomethane while stirring. Stopper the flask and stir at room temperature for 4 h. After the reaction is complete, transfer the reaction solution to a separatory funnel, add 10 times the volume of H2O and extract three times with ethyl acetate. Collect the ethyl acetate phase, remove residual water with anhydrous sodium sulfate, filter, concentrate the organic phase, and purify the crude product by normal-phase silica gel column chromatography (petroleum ether:ethyl acetate = 20:1, v / v) to obtain intermediate T1.

[0034] 2. Preparation of intermediate (4,6-di(dimethylamino)-1,3,5-triazin-2-yl)-L-alanine (T2)

[0035] L-alanine (76.2 mg, 0.855 mmol) and sodium carbonate (157.68 mg, 1.488 mmol) were added to a 25 mL round-bottom flask, and dissolved in 6 mL of H2O using a magnetic stirrer. The flask was fixed on a thermostatically heated magnetic stirrer, and the mixture was stirred and heated to 65 °C. T1 (150 mg, 0.744 mmol) dissolved in 2 mL of anhydrous ethanol was added, and the mixture was heated to 120 °C and refluxed for 12 h. After the reaction was complete, heating was stopped, and the flask was placed in an ice bath to cool. The mixture was purified by reverse extraction with dichloromethane solution. The aqueous phase was retained, and the pH was adjusted to 3-4 with 0.5 M hydrochloric acid aqueous solution. The reaction solution was then extracted five times with dichloromethane in a separatory funnel. The organic phase was collected, dehydrated with anhydrous sodium sulfate, and evaporated to dryness to obtain intermediate T2.

[0036] 3. Preparation of intermediate 4-((4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)-L-alanyl)piperazine-1-carboxylic acid tert-butyl ester (T3)

[0037] Add T2 (165 mg, 0.649 mmol) to a 100 mL round-bottom flask, add a magnetic stir bar, and then add anhydrous acetonitrile (20 mL) to dissolve it. Stir on a flat-plate magnetic stirrer. Add N-methylimidazole (109 μL, 1.363 mmol), then add N-Boc-piperazine (145.6 mg, 0.779 mmol), followed by TCFH (201.1 mg, 0.714 mmol). Stopper the flask and stir at room temperature for 2 h. After the reaction is complete, evaporate the reaction solution to dryness to obtain the crude product, which is purified by normal-phase silica gel column chromatography (dichloromethane:methanol = 100:1, v / v) to obtain intermediate T3.

[0038] 4. Preparation of 4-(N-(4,6-bis(dimethylamino-1,3,5-triazin-2-yl)-N-methyl-L-alanyl)piperazine-1-carboxylic acid tert-butyl ester (T4)

[0039] Add T3 (100 mg, 0.237 mmol), sodium hydride (100 mg, 4.167 mmol), and a magnetic stir bar to a 50 mL round-bottom flask, then add DMF (5 mL). Place the flask on a flat magnetic stirrer and stir. Add iodomethane (0.5 mL) dropwise, stopper the flask, and react for 4 h. After the reaction is complete, place the reaction mixture in a separatory funnel, add 10 times the volume of H2O, and extract three times with ethyl acetate. Collect the organic phase, dry it, and evaporate it to dryness to obtain the crude product. Purify the crude product by normal-phase silica gel column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) to obtain intermediate T4.

[0040] 5. Preparation of (S)-2-((4,6-bis(dimethylamino)-1,3,5-triazin-2-yl)(methyl)amino)-1-(piperazin-1-yl)prop-1-one (TMT-mAla-PP)

[0041] Add T4 (50 mg, 0.115 mmol) to a 25 mL round-bottom flask, dissolve it in dichloromethane (5 mL), add a magnetic stir bar, place the flask on a magnetic stirrer, and add trifluoroacetic acid (0.5 mL) while stirring. React for 4 h. After the reaction is complete, wash the reaction solution three times with saturated NaHCO3 solution, then wash once with H2O. Dry the organic phase with anhydrous sodium sulfate, prepare a sand sample, and purify it by normal-phase silica gel column chromatography (dichloromethane:methanol = 20:1, v / v) to obtain a white final product.

[0042] Structural confirmation: LC-MS / MS: TMT-mAla-PP: [M+H] + =337.22, [M-C4H9N2] + =251.15, [M-C5H9N2O] + =223.15, as Figure 1 As shown. 1 H-NMR (500 MHz, Methanol-d4) δ ppm 5.68 (q, J = 6.8Hz, 1H), 3.74–3.41 (m, 4H), 3.08 (s, 12H), 2.93 (s, 3H), 2.86–2.56 (m, 4H), 1.28 (s, 1H), 1.25 (d, J = 6.7 Hz, 3H), such as Figure 2 As shown.

[0043] Purity determination: Accurately weigh the final product and internal standard (2,4-diamino-6-phenyl-1,3,5-triazine) to prepare an NMR sample, and use... 1 The purity (98.55%) was determined by H-NMR, and the calculation formula is as follows:

[0044] P x =I x × N IS × M x × m IS × P IS / (I IS × N x × M IS × m x ).

[0045] Among them, I x and I ISN is the integrated area of ​​the quantitative peak of the sample and the quantitative peak of the internal standard; x and N IS These represent the number of protons contained in the quantitative peaks of the sample and the internal standard, respectively; M x and M IS These are the relative molecular masses of the sample and the internal standard, respectively; m x and m IS These are the sample weights for the test sample and the internal standard, respectively; P x and P IS These represent the mass fractions of the sample to be tested and the internal standard, respectively.

[0046] Example 2: Use of TMT-mAla-PP as a derivatization reagent for bile acid compounds

[0047] 1. Solution preparation

[0048] Preparation of standard solutions of bile acid compounds: Accurately weigh appropriate amounts of the standards of 6 bile acid compounds, add methanol to prepare a standard stock solution of each bile acid compound with a concentration of 10mM, store at -20℃ for later use, and dilute to the appropriate concentration as needed before use.

[0049] TMT-PP solution: Accurately weigh an appropriate amount of TMT-PP, dissolve it in acetonitrile-methanol (1 / 1, v / v), and quantitatively prepare a TMT-PP solution of the required concentration. Store at -20℃ for later use.

[0050] TMT-Pro-PP solution: Accurately weigh an appropriate amount of TMT-Pro-PP, dissolve it in acetonitrile-methanol (1 / 1, v / v), and quantitatively prepare a TMT-Pro-PP solution of the required concentration. Store at -20℃ for later use.

[0051] TMT-mAla-PP solution: Accurately weigh an appropriate amount of TMT-mAla-PP, dissolve it in acetonitrile-methanol (1 / 1, v / v), and quantitatively prepare a TMT-mAla-PP solution of the required concentration. Store at -20℃ for later use.

[0052] HATU solution: Accurately weigh an appropriate amount of HATU, dissolve it in acetonitrile, and quantitatively prepare a HATU solution of the required concentration. Store at -20℃ for later use.

[0053] 2. Derivatization reaction

[0054] Precisely pipette the standard stock solutions of each of the above bile acid compounds and quantitatively prepare a mixed standard solution of bile acid compounds with a concentration of 0.1 μM using methanol, for later use.

[0055] Accurately pipette 20 μL of the prepared bile acid compound mixed standard solution into a 1.5 mL centrifuge tube, add 40 μL of 6 mM HATU (2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) solution, vortex for 1 min, add 40 μL of 9 mM derivatization reagent (TMT-PP, TMT-Pro-PP, TMT-mAla-PP) solution, vortex for 1 min, centrifuge at low speed for 1 min, and react at 45 °C in a metal bath for 75 min. Centrifuge at low temperature and high speed (14000 rpm, 4 °C, 10 min), and accurately pipette 60 μL of the supernatant to a vial for LC-MS / MS analysis.

[0056] 3. Analysis conditions

[0057] (1) Chromatographic conditions: Column: Agilent Zorbax Eclipse XDB-C18 (2.1×100 mm, 1.8 µm); Injector temperature: 4℃; Flow rate: 0.40 mL / min; Column temperature: 50℃; Mobile phase A: 0.1% formic acid, Mobile phase B: methanol; Gradient: 0-2 min (60-64% B), 2-18 min (64%-69% B), 18-25 min (69%-73% B), 25-30 min (73%-80% B), 30-35 min (80%-83% B), 35-36 min (83%-94% B), 36-41 min (94%-96% B), 41-42 min (96%-100% B), 42-47 min (100% B), 47-47.5 min (100%-60% B), 47.5-53 min (60% B); Injection volume: 3 µL.

[0058] (2) Mass spectrometry conditions: ESI positive ion detection; interface voltage: 4.5 kV; MRM mode; nebulizer (N2) rate: 3.0 L / min; dryer (N2) rate: 15.0 L / min; heating block temperature: 400℃; DL tube temperature: 250℃; Table 1 shows the mass spectrometry detection conditions of the derivatized products obtained by derivatizing 6 bile acid compounds with TMT-PP, TMT-Pro-PP and TMT-mAla-PP as derivatizing reagents.

[0059] Table 1. Mass spectrometry detection conditions for bile acid derivatives

[0060]

[0061] 4. Results

[0062] Using the chemical structures of three similar compounds, TMT-PP, TMT-Pro-PP, and TMT-mAla-PP, as examples... Figure 3 Table 2 shows a comparison of the mass spectrometry response results of the derivatized products obtained by the derivatization reaction of six bile acid compounds with the derivatization reagent.

[0063] Table 2. Mass spectrometry response data of different mass spectrometry probes to the derivatization of six bile acid metabolites.

[0064]

[0065] Table 2 shows that, compared with similar compound TMT-PP, the derivatization products obtained by using TMT-mAla-PP provided in this invention as a derivatization reagent to derivatize six bile acid compounds (including trihydroxy bile acid: CA; dihydroxy bile acids: DCA, UDCA; monohydroxy bile acid: LCA; and glycine-modified bile acids: GCA, GUDCA) have significantly better mass spectrometry response sensitivity (mass spectrometry response improved by about 2.7 to 58 times); compared with similar compound TMT-Pro-PP, the derivatization products obtained by using TMT-mAla-PP provided in this invention as a derivatization reagent to derivatize six bile acid compounds (as before) have significantly better mass spectrometry response sensitivity (mass spectrometry response improved by about 2.8 to 25 times). Figure 4 , Figure 5 , Figure 6 The mass spectrometry peaks of the derivatized products obtained by sequentially derivatizing six bile acid compounds with TMT-PP, TMT-Pro-PP, and TMT-mAla-PP as derivatizing reagents are shown.

[0066] In summary, this invention provides a novel chemical compound, TMT-mAla-PP, and its preparation method. Compared with similar compounds TMT-PP or TMT-Pro-PP in the prior art, the derivatized products obtained by using TMT-mAla-PP provided by this invention as a derivatizing reagent to derivatize bile acid compounds exhibit significantly superior mass spectrometry response sensitivity. Therefore, the compound provided by this invention can be used to prepare derivatizing reagents for bile acid compounds. These reagents can be used to derivatize and modify bile acid compounds before mass spectrometry detection, thereby significantly improving the detection performance of mass spectrometry for bile acid compounds.

[0067] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A compound, characterized in that, The chemical structural formula is as follows: 。 2. A method for preparing the compound according to claim 1, characterized in that, Prepared according to the following synthetic route: 。 3. The preparation method according to claim 2, characterized in that, The preparation method of intermediate T1 includes: adding 6-chloro-2,4-diamino-1,3,5-triazine, sodium hydride and DMF to a reaction vessel, adding iodomethane while stirring, and stirring the reaction at room temperature; after the reaction is completed, the reaction solution is transferred to a separatory funnel, water is added and extracted with ethyl acetate, the ethyl acetate phase is collected, water is removed, the solution is concentrated, and purified by normal phase silica gel column chromatography to obtain intermediate T1.

4. The preparation method according to claim 2, characterized in that, The preparation method of intermediate T2 includes: adding L-alanine and sodium carbonate to a reaction vessel and dissolving them in water; adding intermediate T1 under stirring and heating conditions; heating and refluxing reaction; stopping heating after the reaction is completed; transferring the reaction vessel to an ice bath for cooling; back-extracting with dichloromethane; retaining the aqueous phase; adjusting the pH to 3-4; extracting with dichloromethane; collecting the organic phase; removing water; concentrating to obtain intermediate T2.

5. The preparation method according to claim 2, characterized in that, The preparation method of intermediate T3 includes: adding intermediate T2 to a reaction vessel, dissolving it in anhydrous acetonitrile, and then sequentially adding N-methylimidazole, N-Boc-piperazine, and TCFH, stirring the reaction at room temperature; after the reaction is completed, the reaction solution is evaporated to dryness to obtain the crude product, which is then purified by normal-phase silica gel column chromatography to obtain intermediate T3.

6. The preparation method according to claim 2, characterized in that, The preparation method of intermediate T4 includes: adding intermediate T3, sodium hydride and DMF to a reaction vessel, adding iodomethane dropwise while stirring, and stirring the reaction at room temperature; after the reaction is completed, adding water to the reaction solution and extracting with ethyl acetate, collecting the organic phase, removing water, concentrating, obtaining crude product, and purifying intermediate T4 by normal phase silica gel column chromatography.

7. The preparation method according to claim 2, characterized in that, The preparation method of the target compound TMT-mAla-PP includes: adding intermediate T4 to a reaction vessel, dissolving it in dichloromethane, adding trifluoroacetic acid while stirring, and stirring the reaction at room temperature; after the reaction is completed, the reaction solution is first washed with saturated NaHCO3 solution, then washed with water to remove water from the organic phase, and purified by normal phase silica gel column chromatography to obtain the final product.

8. Use of the compound of claim 1 in the preparation of derivatizing reagents.

9. The use according to claim 8, wherein the derivatizing reagent is used to derivatize the bile acid compound before mass spectrometry detection of the bile acid compound.

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