A compound and its preparation method and use

Abstract

The present application relates to the field of organic compounds, and specifically to a compound and its preparation method and use. It has the following general formula: The compound of the present application is amphiphilic and can be used as a cell membrane probe to observe the fine structure of the cell membrane, which is of great significance for studying the morphological changes of the cell membrane.

Description

Technical Field

[0001] The present application relates to the field of organic compounds, and in particular to a compound and a preparation method and use thereof. Background Art

[0002] The cell membrane is a protective barrier for cells and an important submicroscopic structure in the cell structure. Morphological changes in the cell membrane occur in many disease processes, and observing the morphological changes in the cell membrane is helpful to understand the role of the cell membrane in the development of pathology. However, current commercial probes such as DiD cannot observe the fine structure of the cell membrane due to low brightness, inability to anchor to the cell membrane, easy internalization into the cell, and complex operation (staining takes 10-20 minutes and needs to be washed after staining).

[0003] To this end, the present invention is proposed. Summary of the invention

[0004] The main purpose of the present invention is to provide a compound and a preparation method and use thereof. The compound has amphiphilicity and can be used as a cell membrane probe to observe the fine structure of the cell membrane, which is of great significance for studying the morphological changes of the cell membrane.

[0005] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0006] The first aspect of the present invention provides a compound having the following general formula:

[0007]

[0008] Wherein R1 is selected from - one of CH3;

[0009] R2 is selected from - one of the OH;

[0010] R3 and R4 are each independently selected from an alkyl group of C12 or above;

[0011] The condition is that when R1 is -CH3, R2 is not -OH.

[0012] Compared with existing fluorescent probes, in the compounds provided by the present invention, at least one of R1 and R2 is an amphiphilic group, and the selected group has strong lipophilicity and hydrophilicity, so it can be stably anchored on the cell membrane with similar properties, thereby achieving rapid staining, and has a lower internalization rate and a higher signal-to-background ratio, and can be used for long-term dynamic observation of changes in cell membrane structure, and even imaging of fine cell membrane structures such as pseudopodia, microfilaments, intercellular microtubules and migratory bodies.

[0013] In some embodiments, R3 and R4 are each independently selected from a linear alkyl group of C12 or above. The linear alkyl group has low steric hindrance, thereby ensuring high ultraviolet absorption intensity and fluorescence emission intensity.

[0014] In some embodiments, R3 and R4 are the same. Such compounds are easier to synthesize.

[0015] In some embodiments, R1 is selected from R2 is selected from

[0016] Modifying multiple positions of the conjugated ring with amphiphilic groups can improve the fluorescence intensity, making the results more accurate and the signal-to-background ratio higher when imaging fine structures such as pseudopodia, microfilaments, intercellular microtubules, and migratory bodies.

[0017] In some embodiments, the following compound:

[0018]

[0019] The second aspect of the present invention provides a method for preparing the compound described in the first aspect, which is divided into the following two situations.

[0020] When the R1 is The following steps are included:

[0021] Step a1, synthesizing compound 3 according to the following reaction formula;

[0022]

[0023] Step a2, synthesizing compound C according to the following reaction formula;

[0024]

[0025] And optionally step a3, synthesizing compound E according to the following reaction formula;

[0026]

[0027] When the R1 is -OH, the method comprises the following steps:

[0028] Step b, synthesizing compound F according to the following reaction formula;

[0029]

[0030] In the first case above, whether step a3 is performed depends on the type of R2 in the target compound. When R2 is selected from -OH, step a3 is not required.

[0031] Although the synthetic steps of different compounds are different, the reaction mechanism is the same.

[0032] In some embodiments, the step a1 is carried out in a strong acid environment, and the strong acid preferably includes at least one of concentrated sulfuric acid or trifluoromethanesulfonic acid; the reaction temperature of the step a1 is preferably 120-150°C, more preferably 140°C, and the reaction time is preferably 12h to 24h, more preferably 12h.

[0033] In some embodiments, in step a2, step a3, and step b, an amidating agent and a weak base are used to activate the carboxyl group, and the amidating agent preferably includes 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (abbreviated as HATU); the weak base preferably includes N,N-diisopropylethylamine (abbreviated as DIPEA); the reaction temperature of step a2, step a3, and step b is preferably 20-30°C.

[0034] In some embodiments, the reactions of step a2, step a3 and step b are carried out in an organic solvent, and the organic solvent is preferably N,N-dimethylformamide.

[0035] In some embodiments, the step a1, the step a2, the step a3, and the step b are reacted under nitrogen protection.

[0036] The yield can also be increased by adjusting the amount of reactants, as follows.

[0037] In some embodiments, the molar ratio of 4-diethylaminoketo acid (Compound A) to Compound 1 is 1:1.1;

[0038] In some embodiments, the molar ratio of compound 2 to compound 3 is 1.5:1;

[0039] In some embodiments, in step a2, the molar ratio of compound 3 to the amidation agent is 1:1.2;

[0040] In some embodiments, in step a3, the molar ratio of compound C to compound D is 1:1.2;

[0041] In some embodiments, in step a3, the molar ratio of compound C to the amidation agent is 1:1.2;

[0042] In some embodiments, the molar ratio of compound 4 to compound D is 1:1.5;

[0043] In some embodiments, the molar ratio of compound 4 to the amidation agent is 1:1.2.

[0044] In some embodiments, RMG1, RMG2 and RMG3 are prepared by the following method.

[0045]

[0046] Preparation of RMG2: 4-diethylaminoketoacid and compound 1 are dissolved in a strong acid and heated to obtain compound 3. Compound 3 is dissolved in an organic solvent, and the carboxyl group is activated using an amidation reagent and a weak base, and then compound 2 is added and stirred at room temperature to obtain RMG2.

[0047] Preparation of RMG3: RMG2 was dissolved in an organic solvent and the carboxyl group was activated using an amidation reagent and a weak base, and then compound 2 was added and stirred at room temperature to obtain RMG3.

[0048] Preparation of RMG1: Rhodamine B chloride was dissolved in an organic solvent and the carboxyl group was activated using an amidation reagent and a weak base, and then compound 2 was added and stirred at room temperature to obtain RMG1.

[0049] The third aspect of the present invention provides the use of the compound described in the first aspect as a fluorescent probe anchored to the cell membrane, which can be used for imaging fine structures of the cell membrane such as pseudopodia, microfilaments, intercellular microtubules and migratory bodies.

[0050] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0051] 1) The compound of the present invention can quickly stain the cell membrane within one minute and perform fluorescence imaging without washing.

[0052] 2) The compounds of the present invention have lower internalization rates and higher signal-to-background ratios.

[0053] 3) The compounds of the present invention are modified with lipophilic alkyl long chains and hydrophilic sulfonic acid groups, have good amphiphilicity, and are closer to the chemical properties of cell membranes. Therefore, they can anchor to cell membranes for a long time, and can be used to dynamically observe changes in cell membrane structure for a long time.

[0054] 4) The compounds of the present invention can be used for imaging fine structures of cell membranes such as pseudopodia, microfilaments, intercellular microtubules and migratory bodies.

[0055] 5) The compounds of the present invention can be used as fluorescent probes to achieve imaging of migratory bodies.

[0056] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present application. Moreover, the same reference numerals are used throughout the drawings to represent the same components. In the drawings:

[0058] Figure 1 and 2 They are respectively the ultraviolet absorption intensity and fluorescence emission intensity of the probe RMG1 in the present invention in different organic solvents; the abscissa is the wavelength in each solvent, and the ordinate is the absorption intensity or fluorescence intensity.

[0059] Figure 3 and 4 They are respectively the ultraviolet absorption intensity and fluorescence emission intensity of the probe RMG2 in the present invention in different organic solvents.

[0060] Figure 5 and 6 are respectively the ultraviolet absorption intensity and fluorescence emission intensity of the probe RMG3 in the present invention in different organic solvents; wherein, Figures 1 to 6 The different colored curves correspond to different solvents, and their meanings are the same as Figure 6 The markings are the same.

[0061] Figure 7 The cell membrane fluorescence imaging diagram of the three cell membrane fluorescent probes RMG1, RMG2 and RMG3 in the present invention; the probe concentration is 2 μM; the excitation light source wavelength is 488 nm, and the emission wavelength is collected at 500-600 nm.

[0062] Figure 8 The cell membrane probe RMG3 of the present invention is used for fluorescence imaging of fine cell membrane structures such as pseudopodia, microfilaments, intercellular microtubules and migratory bodies. DETAILED DESCRIPTION

[0063] The following embodiments of the technical solution of the present application will be described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0065] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0066] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0067] The meanings of the abbreviations in this application are as follows:

[0068] DMF: N,N-dimethylformamide. HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate. DIPEA: N-ethyldiisopropylamine. MeOH: methanol. CAN: acetonitrile. DMSO: dimethyl sulfoxide. Water: water. THF: tetrahydrofuran. Dioxane: dioxane. EtOH: ethanol. EA: ethyl acetate.

[0069] Example 1, Preparation method of RMG1, RMG2, RMG3

[0070] The synthetic route is as described above, and the reaction conditions are as follows.

[0071] Synthesis of probe RMG1: 200 mg of rhodamine B chloride was dissolved in 4 mL of ultra-dry DMF solution, and 191 mg of HATU and 87 μL of DIPEA were added under nitrogen protection and stirred at room temperature for two hours to activate the carboxyl group. Then, an ultra-dry DMF solution containing 153 mg of compound 1 and 138 μL of DIPEA was added and stirred at room temperature overnight. After the reaction was stopped the next day, the excess solvent was removed by decompression using a rotary evaporator, and pure dichloromethane was used as an eluent for column chromatography to purify the purple solid powder RMG1 (198 mg, 0.27 mmol, 64%).

[0072] The structure of RMG1 was confirmed as follows:

[0073] 1H NMR (400MHz, CDCl3) δ7.64(d,J=7.0Hz,1H),7.61(d,J=7.7Hz,1H),7.55(t,J=8.4Hz,1H),7.37(d,J=9.3Hz,2H),7.20(d,J=14.4Hz,2H) ,6.88–6.81(m,1H),6.66(s,2H),3.71–3.48(m,12H),3.20(s,2H),2.76(s,2H),1.32(s,20H),1.25(s,12H),0.87(d,J=6.0Hz,3H).13C NMR (101MHz, CDCl3) δ168.31,163.20,157.66,157.52,156.58,155.55,141.63 ,136.69,129.89,127.54,124.39,114.03,113.50,113.39,99.99,97.40,96.7 2,95.73,53.86,50.37,48.92,47.23,46.07,44.83,43.73,43.40,42.60,42.33,42.17,33.45,31.04,29.50,28.52,26.88,25.93,24.59,22.29,18.86,18.68 18.17,17.40,15.12,14.11,13.77,12.68,12.35,12.15.

[0074] HR-ESI-MS: m / z [C 43 H 62 N3O5S], calculated as 732.4405; the actual measured value is 732.4410. m / z[C 43 H 61 N3NaO5S], calculated to be 754.4230; the actual measured value is 754.4227.

[0075] Synthesis of compound 3: 1.1 g of compound 2 and 1.56 g of 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid were dissolved in 15 mL of trifluoromethanesulfonic acid and heated to 150°C for 12 hours. After stopping the reaction and cooling, the reaction solution was poured into 200 mL of ice water and extracted with dichloromethane three times (150 mL×3). Column chromatography was performed with dichloromethane / methanol (100:1 to 10:1) to obtain red solid powder compound 3 (884 mg, 1.76 mmol, 36%).

[0076] The structure of compound 3 was confirmed as follows:

[0077] 1H NMR(400MHz, CDCl3)δ=8.10(s,1H),7.53(s,2H),7.11(s,1H),6.90(s,2H),6.59(s,4H),3.44(s,9H),2.21(s,2H),1.76(s,2H),1.19(s,9H).13C NMR(101MHz, CDCl3)δ=156.11,132.35,131.65,130.15,96.71,77.29,77.03,76.71,51.43, 50.18,45.27,43.22,42.32,37.11,37.11,42.41,33.31,22.77,18.47,12.27,11.72,11.18.

[0078] HR-ESI-MS: m / z [C 30 H 33 N2O5], calculated to be 501.2384; the actual measured value is 501.2383.

[0079] Synthesis of probe RMG2: Under the protection of nitrogen, 238 mg of compound 3, 188 mg of HATU and 160 μL of DIPEA were dissolved in 4 mL of ultra-dry DMF and stirred at room temperature for 10 minutes. Then, ultra-dry DMF containing 152 mg of compound 1 and 160 μL of DIPEA was added dropwise to the above mixture and stirred at room temperature for 24 hours. After stopping the reaction, the solvent was removed using a rotary evaporator, and column chromatography was performed using ethyl acetate / methanol (100:1 to 4:1) to obtain purple solid RMG2 (221 mg, 0.28 mmol, 58%).

[0080] The structure of RMG2 was confirmed as follows:

[0081] 1H NMR (400MHz, MeOD-d4) δ = 8.14 (d, J = 6.3, 1H), 7.63 (dd, J = 9.2, 5.4, 2H), 7.29–7.19 (m, 3H), 7.07 (d ,J=9.6,1H),7.02(s,1H),6.97(dd,J=9.4,2.7,1H),6.93(d,J=2.1,1H),3.72–3.56(m,8H),3.50( dd,J=15.7,10.5,2H),3.40–3.32(m,2H),2.81(dd,J=13.9,7.0,2H),2.52(dt,J=21.7,6.4,2H),2 .01(dd,J=15.4,7.3,4H),1.91(s,1H),1.54(s,2H),1.27(d,J=11.4,28H),0.91-0.85(m,3H).13C NMR(101MHz,MeOD-d4)δ=157.96,155.79,131.72,129.36,113.68,95.68,48.69,48.25,48.04,47.83 ,47.61,47.40,47.19,46.97,45.60,45.29,31.68,29.36,29.08,27.45,26.70,22.34,13.06,11.40.

[0082] HR-ESI-MS: m / z [C 45 H 62 N3O7S], calculated to be 788.4327; the actual measured value is 788.4306.

[0083] Synthesis of probe RMG3: Under nitrogen protection, 250 mg of compound 3 was dissolved in 5 mL of ultra-dry DMF solution and 388 mg of HATU and 330 μL of DIPEA were added. After continuous stirring at room temperature for ten minutes to activate the carboxyl group, 5 mL of ultra-dry DMF containing 313 mg of compound 1 and 330 μL of DIPEA was added to dissolve. After reacting overnight for 24 hours, the liquid was removed using a rotary evaporator, and purple solid RMG3 (248 mg, 0.23 mmol, 46%) was obtained after purification by column chromatography with ethyl acetate / methanol (100:1 to 2:1).

[0084] The structure of RMG3 was confirmed as follows:

[0085] 1H NMR (400MHz, MeOD-d4) δ = 7.74 (t, J = 5.8, 2H), 7.65 (d, J = 7.5, 1H), 7.50 (d, J = 8.3, 1H), 7.32 (t ,J=9.8,2H),7.11(d,J=9.7,2H),7.05(s,1H),6.97(s,1H),3.69(dd,J=14.2,7.0,8H),3.52( s,2H),3.35(s,2H),3.22(s,3H),2.82(d,J=6.0,2H),2.68(t,J=6.8,1H),2.55(dd,J=14.0,6 .5,2H),1.99(d,J=26.3,6H),1.56(s,2H),1.43–1.08(m,46H),0.89(dd,J=13.0,6.2,9H).13C NMR(101MHz,MeOD-d4)δ=157.89,156.09,132.25,130.18,129.35,113.62,95.89,50.74,4 5.52,43.48,31.68,29.30,28.55,27.47,26.64,26.15,23.96,22.36,20.99,13.08,11.55.

[0086] HR-ESI-MS: m / z [C 60 H 93 N4O9S2], calculated to be 1077.6389; the actual measured value is 1077.6393.

[0087] The cell membrane fluorescent probes RMG1, RMG2 and RMG3 prepared by the above method were used in Examples 2 and 3.

[0088] Example 2. Absorption and fluorescence spectra of probes RMG1, RMG2, and RMG3 in different solvents

[0089] Dilute the probes RMG1, RMG2, and RMG3 in solvents of different polarities (such as Figure 1 The final concentration was 2 μM. The absorption spectrum was then measured using a spectrophotometer; the fluorescence spectra were collected using 488 nm as the excitation.

[0090] The results are as follows Figures 1 to 6 As shown, the compound of the present invention has very high ultraviolet absorption intensity and fluorescence emission intensity in various organic solvents, especially in methanol and ethyl acetate EA, and has relatively weak ultraviolet absorption intensity and fluorescence emission intensity in water.

[0091] Real-time example 3, cell membrane imaging effect of probes RMG1, RMG2, and RMG3

[0092] The probes RMG1, RMG2, and RMG3 were diluted to 2 μM using DMEM high-glucose medium without phenol red, and the dilutions were used to rapidly stain HepG2 cells for 1 minute. Then, a fluorescence confocal imager was used with 488 nm as the excitation light source to collect the fluorescence intensity in the 500-600 nm band.

[0093] The results are as follows Figure 2 As shown, the compound of the present invention can show significant fluorescence intensity after 1 minute of staining, and the staining time is greatly shortened compared with the existing compounds.

[0094] Example 4: Imaging of cell membrane fine structure using probe RMG3

[0095] The probe RMG3 was diluted to 2 μM with DMEM high-glucose medium without phenol red, and then stained HepG2 cells for one minute. Then, a fluorescence confocal imager was used with 488 nm as the excitation light source to collect the fluorescence intensity in the 500-600 nm band. The fine structures of the cell membrane, such as pseudopodia, microfilaments, intercellular microtubules, and migratory bodies, were observed.

[0096] The results are as follows Figure 3 As shown, the compound of the present invention can clearly observe fine cell membrane structures such as pseudopodia, microfilaments, intercellular microtubules and migratory bodies.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should all be included in the scope of the claims and specification of the present application. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall within the scope of the claims.

Claims

1. A compound, characterized in that Has the following general formula: Wherein R1 is selected from - one of CH3; R2 is selected from - one of the OH; R3 and R4 are each independently selected from an alkyl group of C12 or above; The condition is that when R1 is -CH3, R2 is not -OH.

2. The compound according to claim 1, characterized in that R3 and R4 are each independently selected from a C12 or higher linear alkyl group.

3. The compound according to claim 1, characterized in that R3 and R4 are the same.

4. The compound according to any one of claims 1 to 3, characterized in that R1 is selected from R2 is selected from 5. The compound according to claim 1, characterized in that For the following compounds:

6. A method for preparing a compound according to any one of claims 1 to 5, characterized in that: When the R1 is The following steps are included: Step a1, synthesizing compound 3 according to the following reaction formula; Step a2, synthesizing compound C according to the following reaction formula; And optionally step a3, synthesizing compound E according to the following reaction formula; When the R1 is -OH, the method comprises the following steps: Step b, synthesizing compound F according to the following reaction formula; 7. The method for preparing the compound according to claim 6, characterized in that: The step a1 is carried out in a strong acid environment.

8. The method for preparing the compound according to claim 7, characterized in that: The strong acid is at least one of concentrated sulfuric acid or trifluoromethanesulfonic acid.

9. The method for preparing the compound according to claim 6, characterized in that: The reaction temperature of step a1 is 120-150°C.

10. The method for preparing the compound according to claim 6, characterized in that: The reaction time is 12h~24h.

11. The method for preparing the compound according to claim 6, characterized in that: In the step a2, the step a3 and the step b, an amidation reagent and a weak base are used to activate the carboxyl group.

12. The method for preparing the compound according to claim 11, characterized in that: The amidation reagent is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

13. The method for preparing the compound according to claim 11, characterized in that: The weak base is N,N-diisopropylethylamine.

14. The method for preparing the compound according to claim 6, characterized in that: The reaction temperature of step a2, step a3 and step b is 20-30°C.

15. The method for preparing the compound according to claim 6, characterized in that: The reactions of step a2, step a3 and step b are carried out in an organic solvent.

16. The method for preparing the compound according to claim 15, characterized in that: The organic solvent is N,N-dimethylformamide.

17. The method for preparing the compound according to claim 6, characterized in that: The step a1, the step a2, the step a3 and the step b are reacted under nitrogen protection.

18. Use of the compound according to any one of claims 1 to 5 for preparing a fluorescent probe anchored to a cell membrane.

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