Light-operated activated covalent probe for transthyretin and synthetic method of light-operated activated covalent probe

Abstract

The invention relates to a light-operated activated covalent probe for transthyretin and a synthesis method of the light-operated activated covalent probe. The probe has a structure as shown in a formula I, the synthesis raw materials are low in price, and the method is simple, easy to operate and easy to purify; the probe can target transthyretin, and can activate and produce carbene under the irradiation of ultraviolet light or white light, so as to carry out covalent labeling on the protein; the method can be applied to the fields of transthyretin quantitative analysis and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of probes. Specifically, the present invention relates to a class of photocontrolled activation covalent probes for transthyretin and a method for synthesizing the same. Background Art

[0002] Transthyretin (TTR) is a plasma tetrameric protein composed of 127 amino acids, mainly responsible for the transport functions of thyroid hormones and vitamin A. Under the action of factors such as gene mutations, environmental stress, and natural aging, the TTR tetramer will dissociate abnormally and misfold, forming fibrous amyloid deposits. These deposits may trigger transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

[0003] Currently, researchers have developed TTR tetramer covalent probes based on the lysine activation mode of pKa perturbation, using chemical reactions such as nucleophilic substitution and Michael addition for labeling, and the reactive groups include 1,4-Michael acceptors, sulfonyl fluorides, and active esters, etc. However, there has been no report on using photocontrolled activation covalent probes for TTR labeling. Compared with traditional covalent probes, photocontrolled activation probes have the advantage of spatio-temporal controllability and can precisely regulate reactions at specific times and locations.

[0004] Therefore, the development of photocontrolled activation TTR covalent labeling probes will provide new research means for the development of TTR tetramer stabilizers and the quantitative analysis of TTR concentration. This has important scientific significance and clinical value for deeply understanding the mechanism of transthyretin amyloidosis diseases and promoting their clinical diagnosis and treatment. Summary of the Invention

[0005] According to one aspect of the present application, there is provided a Compound I, which is a class of photocontrolled activation covalent probes and can covalently label transthyretin. The above properties of the fluorescent molecule can be used for the development of TTR tetramer stabilizers and the research on the quantitative analysis of TTR concentration, etc.

[0006] According to one aspect of the present application, there is provided a Compound I, which has the chemical formula shown in Formula I:

[0007]

[0008] In Formula I:

[0009] R1 is selected from one of C1-C5 alkyl, substituted C1-C5 alkyl, and C3-C8 cycloalkyl;

[0010] R2 is the same or different and each independently selected from the group consisting of hydrogen, halogen, C1-C5 alkyl, C1-C5 alkoxy, substituted C1-C5 alkyl, and hydroxy;

[0011] R3 is selected from the group consisting of hydrogen, hydroxy, amino, -OR4, -NHR4, C1-C5 alkyl, and substituted C1-C5 alkyl;

[0012] R4 is selected from the group consisting of C1-C5 alkyl, substituted C1-C5 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl.

[0013] In one embodiment, the "substituted" means mono- or poly-substituted by deuterium, halogen, or hydroxy.

[0014] In one embodiment, the substituted C1-C5 alkyl includes deuterated C1-C5 alkyl and halogenated C1-C5 alkyl; the substituted C2-C6 alkenyl includes deuterated C2-C6 alkenyl and halogenated C2-C6 alkenyl; the substituted C2-C6 alkynyl includes deuterated C2-C6 alkynyl and halogenated C2-C6 alkynyl.

[0015] In one embodiment, R1 is selected from the group consisting of methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, trifluoromethyl, chloromethyl, bromomethyl, iodomethyl, cyclopropane, cyclobutane, cyclohexane, cycloheptane, and cyclooctane.

[0016] In one embodiment, R2 is the same or different and each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, trifluoromethyl, chloromethyl, bromomethyl, iodomethyl, methoxy, ethoxy, and propoxy.

[0017] In one embodiment, R3 is selected from the group consisting of -OR4 and -NHR4; R4 is selected from the group consisting of methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and pentyl. Wherein, m and n are independently selected from 0, 1, 2, 3, or 4.

[0018] In one embodiment, the compound is selected from:

[0019]

[0020] According to another aspect of the present application, there is also provided a method for preparing the above compound I, comprising the following steps:

[0021]

[0022] In one embodiment, step (1) includes:

[0023] Dissolve the basic substance in solvent I and cool to -5 to 5 °C. Under the protection of an inert gas, add Compound II and stir at -5 to 5 °C for 5 - 60 minutes. Then add Compound III and raise the temperature to 20 - 40 °C and stir. After the reaction is completed, add an acid to adjust to acidic, remove solvent I, add an organic solvent and water for extraction, and separate the organic phase by column chromatography to obtain Intermediate I.

[0024] Preferably, the basic substance is selected from at least one of sodium tert-butoxide, potassium tert-butoxide, sodium methoxide, sodium ethoxide, potassium hydroxide, sodium hydroxide, sodium hydride; the molar ratio of the basic substance to Compound II is 2 - 6:1.

[0025] Preferably, the reaction conditions of step (1) are: temperature is 20 - 30 °C; time is 2 - 12 h.

[0026] Preferably, the molar ratio of Compound II to Compound III is 1:1 - 2.

[0027] Preferably, solvent I is selected from at least one of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, toluene.

[0028] Preferably, the inert gas is selected from at least one of nitrogen, argon.

[0029] Preferably, the acid is selected from at least one of acetic acid, hydrochloric acid, sulfuric acid.

[0030] Preferably, the organic solvent in the extraction is selected from at least one of ethyl acetate, dichloromethane, chloroform, toluene.

[0031] In one embodiment, step (2) includes:

[0032] Dissolve Intermediate I, Compound IV, and 4-dimethylaminopyridine in solvent II and cool to -5 to 5 °C. Subsequently, add dicyclohexylcarbodiimide and raise the temperature to 20 - 40 °C and stir. After the reaction is completed, add an organic solvent and water for extraction, and separate the organic phase by column chromatography to obtain Intermediate II.

[0033] Preferably, the reaction conditions of step (2) are: temperature is 20 - 30 °C; time is 12 - 24 h.

[0034] Preferably, the molar ratio of Intermediate I to Compound IV is 1:1.0 - 3.0.

[0035] Preferably, solvent II is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, N,N-dimethylformamide.

[0036] Preferably, the organic solvent in the extraction is selected from at least one of ethyl acetate, dichloromethane, chloroform, and toluene.

[0037] In one embodiment, step (3) includes:

[0038] Dissolve intermediate II and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in solvent III, cool to -5 to 5 °C, slowly add compound V thereto, raise the temperature to 20 - 40 °C and stir. After the reaction is completed, remove solvent III, add an organic solvent and water for extraction, and perform column chromatography separation on the organic phase to obtain compound I.

[0039] Preferably, the reaction conditions of step (3) are: temperature is 20 - 30 °C; time is 2 - 24 h.

[0040] Preferably, the molar ratio of intermediate II to DBU is 1:2.0 - 6.0.

[0041] Preferably, the molar ratio of intermediate II to compound V is 1:1.0 - 3.0.

[0042] Preferably, the solvent III is selected from at least one of acetonitrile, tetrahydrofuran, dichloromethane, chloroform, and N,N-dimethylformamide.

[0043] Preferably, the organic solvent in the extraction is selected from at least one of ethyl acetate, dichloromethane, chloroform, and toluene.

[0044] The probe of the present invention has the characteristics of targeting transthyretin (TTR) and being activated under ultraviolet light irradiation to achieve covalent labeling, and can be used in the fields of TTR protein quantitative analysis, stabilizer development, related disease research, etc.

[0045] Therefore, the present invention also provides the application of compound I in the preparation of a product for detecting transthyretin.

[0046] The present invention also provides the application of compound I in the preparation of a medicament for diagnosing transthyretin amyloidosis.

[0047] Preferably, the transthyretin amyloidosis includes transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

[0048] The beneficial effects of the present application include but are not limited to:

[0049] (1) The synthetic raw materials provided by the present invention are low-cost, the method is simple and easy to operate, and easy to purify;

[0050] (2) This type of probe can be activated under ultraviolet or white light irradiation to generate a reactive carbene intermediate. In methanol, as the irradiation time prolongs, the absorption value decreases.

[0051] (3) This type of probe can target transthyretin, be activated under ultraviolet or white light irradiation to produce carbene, and then covalently label the protein. It can be applied to fields such as quantitative analysis of transthyretin. Description of the Drawings

[0052] Figure 1 It is the synthetic route diagram of this type of probe.

[0053] Figure 2 It is the 1H NMR spectrum of probe P1 prepared in Example 3.

[0054] Figure 3 It is the 13C NMR spectrum of probe P1 prepared in Example 3.

[0055] Figure 4 It is the change of the absorption spectrum of probe P1 after ultraviolet irradiation in methanol. The abscissa is the wavelength, the ordinate is the absorption value, and the probe concentration is 50 μM.

[0056] Figure 5 It is the fluorescence spectra of probe P1 and transthyretin before and after ultraviolet irradiation. The probe concentration is 50 μM, and the protein concentration is 14.4 μM.

[0057] Figure 6 It is the fluorescence spectra of probe P1 and transthyretin before and after white light irradiation. The probe concentration is 50 μM, and the protein concentration is 14.4 μM.

[0058] Figure 7 It is the gel electrophoresis diagram of probe P1 covalently labeling transthyretin under ultraviolet light irradiation.

[0059] Figure 8 It is the gel electrophoresis diagram of probe P1 covalently labeling transthyretin under white light irradiation.

[0060] Figure 9 It is the mass spectrometry diagram of probe P1 covalently labeling transthyretin. Detailed Embodiments

[0061] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Without departing from the spirit and essence of the present invention, simple modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention; if not otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0062] Unless otherwise specified, the raw materials and reagents used in this application are all commercially purchased and used directly without treatment. The instruments and equipment used adopt the schemes and parameters recommended by the manufacturers.

[0063] The NMR data in the examples of this application were obtained using a nuclear magnetic resonance spectrometer Bruker AVANCE III 700MHz;

[0064] The matrix-assisted ionization mass spectrometry model used in the examples of this application is Bruker Ultra Flex III MALDI-TOF-TOF.

[0065] Example 1

[0066] Potassium tert-butoxide (491 mg, 5.0 mmol) was added to tetrahydrofuran (10 mL) and cooled to 0 °C. Under nitrogen protection, compound II (500 mg, 1.7 mmol) was added and stirred at 0 °C for 10 minutes. Subsequently, compound III (345 mg, 1.8 mmol) was added thereto. After stirring at room temperature overnight, acetic acid was added to adjust to acidity. After most of the tetrahydrofuran was removed by rotary evaporation, an appropriate amount of ethyl acetate and water were added for extraction. The organic phase components were collected and concentrated. Finally, it was purified by column chromatography (gradient elution of petroleum ether / ethyl acetate with a volume ratio of 100:0 - 50:50) to obtain intermediate I (257 mg, yield: 48%).

[0067] Intermediate I was tested using a nuclear magnetic resonance spectrometer, and the results are as follows: 1 H-NMR(400MHz,CDCl3)δ7.46(d,J=7.9Hz,2H),7.28(s,2H),7.17(s,2H),6.99(s,2H),4.52(m,2H),3.66(s,2H),2.51(m,1H),2.34(s,6H).

[0068] The synthetic route of this example is as follows:

[0069]

[0070] Example 2

[0071] Intermediate I (83 mg, 0.26 mmol), Compound IV (9.6 mg, 0.3 mmol), and 4-dimethylaminopyridine (173 mg, 0.75 mmol) were dissolved in dichloromethane (5 mL) and cooled to 0 °C. Subsequently, dicyclohexylcarbodiimide (155 mg, 0.75 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, an appropriate amount of water and dichloromethane were added for extraction, and the organic phase components were collected and concentrated. Finally, it was purified by column chromatography (gradient elution with petroleum ether / ethyl acetate with a volume ratio of 100:0 - 50:50) to obtain Intermediate II (32 mg, yield: 37%).

[0072] Intermediate II was tested using a nuclear magnetic resonance spectrometer, and the results are as follows: 1 1H-NMR (400 MHz, CDCl3) δ 7.45 (d, J = 7.9 Hz, 2H), 7.26 (m, 2H), 7.17 (s, 2H), 6.99 (s, 2H), 4.52 (m, 2H), 3.70 (s, 3H), 3.63 (s, 2H), 2.52 (s, 1H), 2.34 (s, 6H).

[0073] The synthesis route of this example is as follows:

[0074]

[0075] Example 3

[0076] Intermediate II (67 mg, 0.2 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (91.3 mg, 0.6 mmol) were dissolved in acetonitrile and cooled to 0 °C. Subsequently, Compound V (52.9 mg, 0.22 mmol) was slowly added thereto, and the mixture was stirred at room temperature overnight. After the reaction was completed, most of the acetonitrile was removed by rotary evaporation. Subsequently, an appropriate amount of water and ethyl acetate were added for extraction, and the organic phase components were collected and concentrated. Finally, it was purified by column chromatography (gradient elution with petroleum ether / ethyl acetate with a volume ratio of 100:0 - 50:50) to obtain the yellow product P1 (62 mg, yield: 65%).

[0077] P1 was tested using a nuclear magnetic resonance spectrometer, and the results are as follows: 1 1H-NMR (400 MHz, CDCl3) δ 7.55 - 7.43 (m, 4H), 7.18 (d, J = 1.9 Hz, 2H), 6.98 (d, J = 2.2 Hz, 2H), 4.52 (s, 2H), 3.88 (d, J = 2.0 Hz, 3H), 2.52 (m, 1H), 2.35 (m, 6H). 1313C-NMR (100 MHz, CDCl3) δ 135.3, 133.5, 131.6, 128.2, 127.2, 127.1, 127.1, 124.2, 79.4, 75.2, 60.0, 52.2, 16.8.

[0078] The synthesis route of this example is as follows:

[0079]

[0080] Test Example 1: Absorption Spectrum Measurement of Probe P1 after Irradiation with Light in Methanol

[0081] Dissolve P1 prepared in the example in methanol (50 μM), irradiate it with ultraviolet light of 360 - 375 nm, and use a microplate reader to measure its absorption spectrum at 0, 5, 20, 40, and 60 minutes. The results are as Figure 4 shown. As the irradiation time of light increases, the absorption value of P1 gradually decreases, indicating that ultraviolet light can activate P1.

[0082] Test Example 2: Fluorescence Spectrum of P1 Covalently Labeled with Transthyretin

[0083] Incubate probe P1 (50 μM) with TTR (14.4 μM) at room temperature for 120 minutes, then irradiate it with ultraviolet light of 360 - 375 nm or white light of 400 - 760 nm for 30 minutes, and perform fluorescence spectrum measurement with an excitation wavelength of 341 nm. The results are as Figure 5 and Figure 6 shown. After P1 is covalently labeled with transthyretin, the fluorescence intensity increases.

[0084] Test Example 3: Fluorescent Gel of P1 Covalently Labeled with Transthyretin

[0085] Incubate probe P1 (50 μM) with TTR (14.4 μM) at room temperature for 120 minutes. After irradiating with ultraviolet light of 360 - 375 nm or white light of 400 - 760 nm for 60 minutes, perform a copper-catalyzed click chemical reaction (50 mM CuSO4, 50 mM TMR-N3, 50 mM TCEP, 1.7 mM TBTA), and react at room temperature for 1 h. Precipitate with acetone overnight, centrifuge, and discard the supernatant. Add 4% SDS solution, 5x loading buffer, denature at 95 °C, and perform gel electrophoresis analysis after cooling. The experimental results are as Figure 7 and Figure 8 shown. Under ultraviolet irradiation conditions, it can be observed that transthyretin is covalently labeled.

[0086] Test Example 4: Mass Spectrum of P1 Covalently Labeled with Transthyretin

[0087] The probe P1 (300 μM) was incubated with TTR (14.4 μM) at room temperature for 120 minutes. After irradiation with ultraviolet light at 360 - 375 nm for 120 minutes, it was diluted with deionized water, and then matrix-assisted laser desorption ionization analysis was carried out. The experimental results are as Figure 9 shown. After transthyretin was covalently labeled with P1, a peak with a molecular weight increase of 320.2 was observed.

[0088] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A compound I having the chemical formula shown in Formula I: In Formula I: R1 is selected from one of C1-C5 alkyl, substituted C1-C5 alkyl, and C3-C8 cycloalkyl; R2 are the same or different and are each independently selected from one of hydrogen, halogen, C1-C5 alkyl, C1-C5 alkoxy, substituted C1-C5 alkyl, and hydroxy; R3 is selected from one of hydrogen, hydroxy, amino, -OR4, -NHR4, C1-C5 alkyl, and substituted C1-C5 alkyl; R4 is selected from one of C1-C5 alkyl, substituted C1-C5 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl.

2. The compound I according to claim 1, wherein The R1 is selected from methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, trifluoromethyl, chloromethyl, bromomethyl, iodomethyl, cyclopropane, cyclobutane, cyclohexane, cycloheptane, and cyclooctane.

3. The compound I according to claim 1, characterized in that, The R2 are the same or different and are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, trifluoromethyl, chloromethyl, bromomethyl, iodomethyl, methoxy, ethoxy, and propoxy.

4. The compound I according to claim 1, wherein R3 is selected from one of -OR4 and -NHR4; R4 is selected from methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, wherein m and n are independently selected from 0, 1, 2, 3 or 4.

5. The compound I according to claim 1, wherein, The compound is selected from:

6. The preparation method of compound I according to any one of claims 1-5, comprising the following steps:

7. According to the preparation method described in claim 6, characterized in that Step (1) includes: dissolving the basic substance in solvent I and cooling to -5 to 5 °C, adding compound II under the protection of inert gas and stirring at -5 to 5 °C for 5-60 minutes, then adding compound III and raising the temperature to 20-40 °C for stirring. After the reaction is completed, adding acid to adjust to acidic, removing solvent I, adding organic solvent and water for extraction, and separating the organic phase by column chromatography to obtain intermediate product I; Step (2) includes: dissolving intermediate product I, compound IV, and 4-dimethylaminopyridine in solvent II and cooling to -5 to 5 °C, then adding dicyclohexylcarbodiimide and raising the temperature to 20-40 °C for stirring. After the reaction is completed, adding organic solvent and water for extraction, and separating the organic phase by column chromatography to obtain intermediate product II; Step (3) includes: dissolving intermediate product II and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in solvent III, cooling to -5 to 5 °C, slowly adding compound V thereto and raising the temperature to 20-40 °C for stirring. After the reaction is completed, removing solvent III, adding organic solvent and water for extraction, and separating the organic phase by column chromatography to obtain compound I.

8. The application of compound I according to any one of claims 1-5 in the preparation of a product for detecting transthyretin.

9. The application of compound I according to any one of claims 1-5 in the preparation of a product for diagnosing transthyretin amyloidosis.

10. The application according to claim 9, characterized in that, The transthyretin amyloidosis includes transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

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