CN110272434B - High-sensitivity acid-base probe, preparation and biological detection application thereof - Google Patents
High-sensitivity acid-base probe, preparation and biological detection application thereof Download PDFInfo
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Abstract
The invention relates to a high-sensitivity acid-base probe, and preparation and biological detection application thereof. The high-sensitivity acid-base probe has a structure shown in the following formula I. The high-sensitivity acid-base probe can be used for measuring tiny pH fluctuation (0.1-0.2 pH) in a solution or a cell biological system, can be used for reversing pH gradient imaging of cancer tissues, selectively lightening the intercellular substance of the cancer tissues, effectively distinguishing the cancer tissues from normal tissues, and is used for diagnosis of cancer parts and auxiliary fluorescence surgery treatment.
Description
Technical Field
The invention relates to a high-sensitivity acid-base probe, and preparation and biological detection application thereof.
Background
The eukaryotic cell contains various highly-differentiated membrane-wrapped subcellular organelle structures, the relative independence between the subcellular organelle structures ensures the optimal environment required by the respective functions, and meanwhile, the energy can be stored in the form of transmembrane potential gradient. In this process, protons (or proton balance) play a very critical role. For example, almost all protein structures and functions are regulated by pH, and the process of protonation-deprotonation predicts a change in the surface charge of an organism and is an integral part of many metabolic reactions. Proton driving force is also critical for cellular energy production and transformation, so intracellular pH is always tightly regulated and varies among subcellular organelles.
Numerous proteins with important physiological activities are highly sensitive to small changes in their surrounding pH, and changes in pH from 0.2 to 0.3 units can greatly affect the function of the organism. For example Na +/ H + Over-activation of the transporter (NHE 1) increases intracellular pH by 0.2-0.7 pH units, inducing transformation events such as cell proliferation. Apoptosis is also accompanied by a slight acidification of cytoplasmic pH, approximately 0.3-0.4 pH units, which is both an important physiological feature of normal tissues and a target for many therapies. Dysregulation of cytoplasmic pH and subcellular organelle pH can affect gene expression, induce protein folding, affect enzyme activity, cause metabolic disorders andgenetic disturbance and the like, and finally various diseases occur.
Dysregulated pH has become an important marker for most solid tumors. Due to the increased expression and activity of transporters on the plasma membrane of cancer cells, especially efflux H + Thus, in contrast to the case of normal extracellular base-internal acids, the intracellular pHi of cancer cells is 7.4 or more, while the extracellular pH is e Is 6.7-7.1. This reversed pH gradient provides a hotbed for the metastasis of cancer cells, the elevated pH i Promoting cancer cell proliferation and apoptosis escape, accelerating cancer cell metabolism adaptability and invasion to organism, and accelerating cancer cell metastasis. Since pH dysregulation is a common feature of cancer, this intracellular and extracellular reversed pH i /pH e The gradient can be used as a marker for cancer diagnosis and a target for cancer treatment.
Since accurate pH regulation inside and outside cells is very important for various cellular functions and physiological processes, accurate monitoring of cellular pH is of great importance. Compared with means of nuclear magnetic resonance or microelectrode pH measurement, the fluorescence microscopy non-invasive real-time detection has great advantages, can accurately measure the pH disturbance of living tissues with high space-time resolution, and provides very important information for researching the physiological and pathological processes of organisms. The small molecular fluorescent probe, the high molecular polymer, the nano material and the green fluorescent protein are more applied fluorescent means, wherein the small molecular fluorescent probe has the advantages of abundant structure, convenient modification, small size, convenient adjustment and capability of entering cells in a free diffusion mode, thereby receiving wide attention.
Although a large number of small-molecule fluorescent probes are applied to the measurement of pH in organisms, the acid-base titration curves of the probes meet the classic acid-base equilibrium Henderson-Hasselbalch equation, but the response range of the probes is 2pH units, and the small pH disturbance cannot be identified and monitored efficiently in real time. Under the condition of the same detection condition, the Hill-type probe can give more obvious signal response to tiny pH change, so that the measurement is more accurate, and the Hill-type probe has important potential in both basic research of biomedicine and clinical application.
Disclosure of Invention
In a first aspect, the present invention provides a compound having the structure of formula I:
in the formula (I), the compound is shown in the specification,
R 1 and R 2 Each independently of the other being H and C1-C6 alkyl, or R 1 And R 2 Linked together as a C2-C6 alkylene group;
R 3 and R 4 Each independently of the other being H and C1-C6 alkyl, or R 3 And R 4 Linked together as a C2-C6 alkylene group;
R 5 and R 6 Each independently is H and C1-C6 alkyl;
R 7 is H, halogen, -CN, -NO 2 C1-C6 alkyl, OR 8 Or NR a R b Wherein R is 8 、R a And R b Each independently of the other being H and C1-C6 alkyl.
In one or more embodiments, R 1 And R 2 Are each C1-C4 alkyl.
In one or more embodiments, R 3 And R 4 Are all C1-C4 alkyl.
In one or more embodiments, R 1 And R 2 Joined together as C2-C4 alkylene, R 3 And R 4 Are linked together as a C2-C4 alkylene group.
In one or more embodiments, R 5 And R 6 Are each C1-C4 alkyl.
In one or more embodiments, R 7 Is NR a R b Wherein R is a And R b Are each C1-C4 alkyl.
In one or more embodiments, R 8 Is a C1-C4 alkyl group.
In one or more embodiments, R 1 And R 2 Are each C1-C4 alkyl; r 3 And R 4 Are each C1-C4 alkyl; and R 5 And R 6 Are all C1-C4 alkyl.
In one or more embodiments, R 1 And R 2 Are each C1-C4 alkyl; r is 3 And R 4 Are each C1-C4 alkyl; and R 5 And R 6 Are each C1-C4 alkyl; r 7 Is H, halogen, -CN, -NO 2 C1-C6 alkyl, OR 8 Or NR a R b (ii) a Wherein R is 8 Is H or C1-C4 alkyl, R a And R b Are each H or C1-C4 alkyl.
In one or more embodiments, R 1 And R 2 Are each C1-C4 alkyl; r 3 And R 4 Are each C1-C4 alkyl; and R 5 And R 6 Are each C1-C4 alkyl; r 7 Is NR a R b (ii) a Wherein R is a And R b Are each C1-C4 alkyl.
In certain embodiments, the compounds of formula I of the present invention are selected from:
the invention also provides a composition comprising a compound described herein.
In one or more embodiments, the composition is a PBS solution or DMSO aqueous solution of the compound.
In one or more embodiments, the composition is a cell culture medium containing the compound.
The present invention also provides a process for the preparation of a compound of formula I as described herein, said process comprising the step of reacting a compound of formula II as described herein with a compound of formula III below in a solvent at 0 ℃ in a solvent:
in the formula III, R 5 And R 6 Each independently is H and C1-C6 alkyl; r is 7 Is H, halogen, -CN, -NO 2 C1-C6 alkyl, OR 8 Or NR a R b Wherein R is 8 、R a And R b Each independently is H and C1-C6 alkyl;
the invention also provides a kit containing a compound or composition described herein.
The invention also provides the use of a compound of formula I as described herein for the manufacture of a reagent or kit for monitoring pH fluctuations in a subject in real time or for cancer diagnosis.
Drawings
FIG. 1: (a) the ultraviolet-visible absorption spectrum of PHX6 at different pH values; (b) Absorbance at 533nm at different pH values (■) and a function non-linear fit curve of dose response (pink); (c) fluorescence emission spectra of PHX6 at different pH values; (d) Absorbance at 575nm at different pH values (■) and a function non-linear fit curve of dose response (pink); all data were collected from 10. Mu.M DMSO-containing aqueous solutions of the probes.
FIG. 2 is a schematic diagram: (a) the uv-vis absorption spectra of PHX1 at different pH values; (b) Absorbance at 533nm at different pH values (■) and a biphasic dose response function non-linear fit curve (pink); (c) fluorescence emission spectra of PHX1 at different pH values; (d) Absorbance at 575nm at different pH values (■) and a biphasic dose response function non-linear fit curve (pink); the inset shows the Hill component (blue curve) and HH component (green curve). All data were collected from 10. Mu.M DMSO-containing aqueous solutions of the probes.
FIG. 3: (a) the ultraviolet-visible absorption spectrum of PHX2 at different pH values; (b) Absorbance at 533nm at different pH values (■) and a biphasic dose response function non-linear fit curve (pink); (c) fluorescence emission spectra of PHX2 at different pH values; (d) Absorbance at 575nm at different pH values (■) and a biphasic dose response function non-linear fit curve (pink); the inset shows the Hill component (blue curve) and HH component (green curve). All data were collected from 10. Mu.M DMSO-containing aqueous solutions of the probes.
FIG. 4 is a schematic view of: (a) the uv-vis absorption spectra of PHX3 at different pH values; (b) Absorbance at 533nm at different pH values (■) and a biphasic dose response function nonlinear fit curve (pink); (c) fluorescence emission spectra of PHX3 at different pH values; (d) Absorbance at 575nm at different pH values (■) and a biphasic dose response function nonlinear fit curve (pink); the inset shows the Hill component (blue curve) and HH component (green curve). All data were collected from 10. Mu.M DMSO-containing aqueous solutions of the probes.
FIG. 5: (a) the uv-vis absorption spectrum of PHX4 at different pH values; (b) Absorbance at 533nm at different pH values (■) and dose response function non-linear fit curve (pink); (c) fluorescence emission spectra of PHX4 at different pH values; (d) Absorbance at 575nm at different pH values (■) and dose response function non-linear fit curve (pink); all data were collected from 10. Mu.M DMSO-containing aqueous solutions of the probes.
FIG. 6: (a) the uv-vis absorption spectrum of PHX5 at different pH values; (b) Absorbance at 533nm at different pH values (■) and dose response function non-linear fit curve (pink); (c) fluorescence emission spectra of PHX5 at different pH values; (d) Absorbance at 575nm at different pH values (■) and dose response function non-linear fit curve (pink); all data were collected from 10. Mu.M DMSO-containing aqueous solutions of the probes.
FIG. 7: cell co-localization experiments of Hela cells. (a-c) bright field, PHX2 fluorescence (. Lamda.) ex =559nm and λ em =570-630 nm) and a, b superimposed pictures; (d-f) 0.5. Mu.M LysoSensor Green DND-189 (Green, lambda ex ＝405nm,λ em =460-530 nm), 0.5 μ M PHX2 (red) and superimposed pictures of d, e; (g-i) rhodamine 123 (Green, λ) ex ＝405nm,λ em =460-530 nm), 0.5 μ M PHX2 (red) and g, h overlay pictures. Scale bar: 10 μm.
FIG. 8: hepa1-6 cell fluorescence imaging.
Red AM (HH-type, line A, 5. Mu.M), PHX1 (Hill-type, line B, 15. Mu.M), PHX3 (Hill-type, line C, 15. Mu.M) and PHX2 (Hill-type, line D, 15. Mu.M). pH =7.2,6.9,6.7,6.4,6.1,5.8,5.5,5.2,5.0. Scale bar: 50 μm. Lambda [ alpha ] ex ＝559nm,λ em =565-640nm the lowest colored band shows the intensity of fluorescence, the yellow is the strongest and the violet is the weakest. (error bars represent standard deviations from 18 different cells selected in six different fields of view).
FIG. 9: a) Pathological images of excised tumor-bearing mouse livers. (B) Fixed living tissue of tumor and (C) Normal liver tissue H&E histopathology images. Confocal fluorescence images of in vitro mouse tumor (D) and normal liver tissue (E) were stained with PHX2 (15. Mu.M) for 15 min. Mean fluorescence intensity of nuclear, cytoplasmic and extracellular regions of tumor (F) and normal tissue (G). Confocal image acquisition lambda ex =559nm and λ em =590-640nm (18 different ROIs from 6 representative image fields; error bars represent variance s.d.).
Detailed Description
It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described below (e.g., the examples) may be combined with each other to constitute a new technical solution.
The high-sensitivity acid-base response probe designed by the invention can be used for measuring tiny pH fluctuation (0.1-0.2 pH) in a solution or a cell biological system, can be used for reversing pH gradient imaging of cancer tissues, selectively lights the intercellular substance of the cancer tissues, effectively distinguishes the cancer tissues from normal tissues, and is used for cancer part diagnosis and auxiliary fluorescence surgery treatment.
In the present invention, alkyl includes straight-chain and branched alkyl groups, generally 1 to 6 carbon atoms in length, preferably 1 to 4 carbon atoms. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (Pr), n-butyl, isobutyl, tert-butyl, and the like.
In the present invention, halogen means fluorine, chlorine, bromine and iodine, and usually fluorine, chlorine and bromine.
The present invention provides compounds of formula I:
in the formula (I), the compound is shown in the specification,
R 1 and R 2 Each independently of the other being H and C1-C6 alkyl, or R 1 And R 2 Linked together as a C2-C6 alkylene group;
R 3 and R 4 Each independently of the other being H and C1-C6 alkyl, or R 3 And R 4 Linked together as a C2-C6 alkylene group;
R 5 and R 6 Each independently is H and C1-C6 alkyl;
R 7 is H, halogen, -CN, -NO 2 C1-C6 alkyl, OR 8 Or NR a R b Wherein R is 8 、R a And R b Each independently of the other being H and C1-C6 alkyl.
In preferred compounds, R 1 And R 2 Are all C1-C4 alkyl. In preferred compounds, R 3 And R 4 Are each C1-C4 alkyl. In preferred compounds, R 5 And R 6 Are each C1-C4 alkyl. In preferred compounds, R 7 Is NR a R b Wherein R is a And R b Are each C1-C4 alkyl. In preferred compounds, R 8 Is C1-C4 alkyl.
In certain embodiments, R 1 And R 2 Joined together as C2-C4 alkylene, R 3 And R 4 Are linked together as a C2-C4 alkylene group.
In certain embodiments, in the compounds of formula I, R 1 And R 2 Are each C1-C4 alkyl; r 3 And R 4 Are each C1-C4 alkyl; and R 5 And R 6 Are each C1-C4 alkyl.
In certain embodiments, in the compounds of formula I, R 1 And R 2 Are each C1-C4 alkyl; r 3 And R 4 Are each C1-C4 alkyl; and R 5 And R 6 Are each C1-C4 alkyl; r 7 Is H, halogen, -CN, -NO 2 C1-C6 alkyl, OR 8 Or NR a R b (ii) a Wherein R is 8 Is C1-C4 alkyl, R a And R b Are each C1-C4 alkyl.
The compounds of formula I can be synthesized using the following general synthetic routes:
first, compound s1 was reacted with Br in ice bath 2 (2.2 equiv.) of the dichloromethane solution was stirred well for 30 minutes. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane and separated on a column. The developer may be 10:1 petroleum ether: ethyl acetate to obtain compound s2.
Next, the compound s2 was dissolved in dry tetrahydrofuran, and then the solution was placed at-78 ℃ and a n-butyllithium solution (2.5 equivalents) was slowly dropped thereinto, followed by sufficiently stirring. After 30 minutes of reaction, a tetrahydrofuran solution of III was slowly added dropwise to the reaction flask. When R in the formula I 7 Is OR 8 Or NR a R b When and R is 8 Or R a Or R b In the case of H atoms, the equivalent of III needs to be reduced. After stirring for 2 hours, the reaction temperature was raised to room temperature. Then adding saturated ammonium chloride solution to quench the reaction, extracting with dichloromethane, and separating and purifying the product by a column. The developer may be 100:1 dichloromethane: methanol.
In the above formula, R 1 -R 8 、R a And R b As defined hereinbefore.
In certain embodiments, the present invention also provides a compound represented by formula II below:
in the formula, R 1 -R 4 In any of the embodiments herein for R in formula I 1 -R 4 The same definition is applied.
For example, in certain embodiments of the compounds of formula II, R 1 And R 2 Each independently of the other being H and C1-C6 alkyl, or R 1 And R 2 Linked together as a C2-C6 alkylene group; r is 3 And R 4 Each independently of the other being H and C1-C6 alkyl, or R 3 And R 4 Are linked together as a C2-C6 alkylene group. Preferred compounds of formula II are those wherein R 1 And R 2 Are each C1-C4 alkyl. Preferred compounds of formula II are those wherein R 3 And R 4 Are each C1-C4 alkyl.
In certain embodiments of formula II, R 1 And R 2 Joined together as C2-C4 alkylene, R 3 And R 4 Are linked together as a C2-C4 alkylene group. In certain embodiments of formula II, R 1 And R 2 Are each C1-C4 alkyl; and R 3 And R 4 Are all C1-C4 alkyl.
The compounds of formula II of the present invention are useful in the preparation of compounds of formula I.
The present invention also provides a process for the preparation of a compound of formula I, said process comprising: a step of reacting a compound of formula II as described herein with a compound of formula III below in a solvent at 0 ℃ or below:
in the formula III, R 5 And R 6 Each independently is H and C1-C6 alkyl; r 7 Is H, halogen, -CN, -NO 2 C1-C6 alkyl, OR 8 Or NR a R b Wherein R is 8 、R a And R b Each independently of the other being H and C1-C6 alkyl.
In certain embodiments, the solvent may be THF, et 2 O, dioxane or any mixed solvent of the solvents.
In certain embodiments, the reaction temperature is from 0 ℃ to-78 ℃.
In certain embodiments, the reaction time is from 1 to 5 hours.
In certain embodiments, the molar ratio of the compound of formula II to the compound of formula III is between 1:1 and 1, 0.5.
In certain embodiments, the reaction temperature is raised to room temperature after the reaction is complete, and then the reaction is quenched by addition of saturated ammonium chloride solution.
In certain embodiments, the product is purified by column separation after extraction with an organic solvent such as dichloromethane. The developer may be a conventional developer such as 100:1 dichloromethane: methanol.
Also provided herein is a composition comprising a compound of formula I herein. The composition may be a solution, such as an aqueous solution or a PBS solution or a DMSO aqueous solution, in which the compound of formula I herein is dissolved, or a medium containing the compound of formula I herein. The culture medium may be any cell culture medium known in the art that can be used for the culture of cells of interest, such as cell culture media used for the culture of Hela cells or Hepa cells. The concentration of the compounds of formula I herein in the composition can be readily determined depending on the particular use of the composition.
Also provided herein is a method of monitoring pH fluctuations comprising the steps of incubating a compound of formula I of the invention with a subject of interest and then reading the intensity of the fluorescence produced. Generally, the object of interest may be a solution or a cell of interest. For example, 0.1 to 15. Mu.M of a compound of formula I of the invention may be contacted with the cell of interest. The cell of interest may be any cell, in particular an animal cell, more particularly a mammalian cell, such as a human cell, e.g. a cell of a diseased tissue or a cell of a normal tissue. After 1-30 minutes of co-incubation, excess free compound is washed away and the fluorescence intensity is read using a conventional fluorescence microscope such as a confocal microscope. For cells, the fluorescence intensity in the cytoplasm, nucleus, and other subcellular organelles, such as mitochondria, lysosomes, etc., can be read. In certain embodiments, after reading the obtained fluorescence intensity, the corresponding pH value is read against a pH and fluorescence intensity titration standard curve to monitor pH differences and pH perturbations. In certain embodiments, the monitoring method is not a disease diagnostic method.
As demonstrated in the examples section, the compounds of formula I of the present invention, in particular PHX2, can be used for reverse pH imaging of cancerous tissue, effectively distinguishing cancerous from normal tissue. Therefore, the invention also includes a method for diagnosing tumors, which comprises contacting the compound of formula I of the present invention with a tissue of interest, and then performing imaging observation, wherein if the intercellular fluorescence intensity is higher than the intracellular fluorescence intensity, the tissue is determined as tumor tissue, otherwise, the tissue is determined as normal tissue. In this method, a solution of a compound of the formula I according to the invention, in particular a PHX2 solution, is usually prepared in an amount of 0.1 to 15. Mu.M, 5 to 15. Mu.M, applied to an unfixed section of biological tissue and the section is visualized by fluorescence microscopy after 5 to 15 minutes. In general, if the intercellular substance fluorescence intensity is significantly higher than the intracellular fluorescence intensity (e.g., 1-fold or more, preferably 2-fold or more, and more preferably 3-fold or more), the tissue can be determined as a tumor tissue; on the contrary, if the intercellular fluorescence intensity is lower than the intracellular fluorescence intensity, the tissue can be determined as a normal tissue. The method can be used for the auxiliary diagnosis of cancer.
Therefore, the invention also provides the use of the compounds of formula I according to the invention for the preparation of a reagent for monitoring pH fluctuations in real time or for cancer diagnosis.
In certain embodiments, the invention also provides a test kit which may contain a compound of the invention or a composition thereof. The test kit may also contain other suitable components depending on its use, such as instruments and/or reagents for preparing tissue sections, etc. The test kit of the invention may be used for the uses described herein, including monitoring pH fluctuations or cancer diagnosis in a subject.
The present invention will be illustrated below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. Unless otherwise indicated, reagents used in the examples and methods employed are those conventional in the art.
Example 1: preparation of compound PHX1
Compound PHX1 was prepared according to the following scheme:
specifically, the compound s1 is first placed on iceUnder bath with Br 2 (2.2 equiv.) of the dichloromethane solution was stirred well for 30 minutes. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane and separated on a column. The developer may be 10:1 petroleum ether: ethyl acetate to give the compound s2 in yield>90 percent. Next, the compound s2 was dissolved in dry tetrahydrofuran, and then the solution was placed at-78 ℃ and a n-butyllithium solution (2.5 equivalents) was slowly dropped thereinto, followed by sufficiently stirring. After 30 minutes of reaction, a tetrahydrofuran solution of III was slowly added dropwise to the reaction flask. After stirring for 2 hours, the reaction temperature was raised to room temperature. Then adding saturated ammonium chloride solution to quench the reaction, extracting with dichloromethane, and separating and purifying the product by a column. The developer may be 100:1 dichloromethane: methanol. 121mg of compound PHX1 are obtained as a red solid in 52% yield.
1 H NMR(400MHz,CDCl 3 ):δ7.22(d,J＝8.7Hz,2H),6.95(d,J＝8.7Hz,1H),6.75(d,J＝2.9Hz,1H),6.71(dd,J＝8.7Hz,3.0Hz,1H),6.42(d,J＝2.5Hz,2H),6.37(dd,J＝8.7Hz,2.6Hz,2H),3.33(q,J＝7.0Hz,8H),2.92(s,6H),2.12(s,2H),1.19(s,6H),1.15(t,J＝7.0Hz,12H)； 13 C NMR(101MHz,CDCl 3 )δ152.7,148.1,147.6,145.7,131.9,126.8,117.7,116.7,114.1,112.1,107.5,99.0,72.4,52.5,44.6,42.2,32.0,31.7,12.7.；ESI-MS(m/z):[M+H] + C 33 H 44 N 3 O 2 Calculated, 514.3434; found 514.3435.
Example 2: preparation of compound PHX2
The compound PHX2 represented by the following formula (128 mg, yield 58%) was obtained as a pale pink solid by a similar method to example 1.
1 H NMR(400MHz,CDCl 3 ):δ7.24(d,J＝8.9Hz,2H),6.93(d,J＝8.7Hz,1H),6.70(s,1H),6.67(d,J＝8.8Hz,1H),6.41(s,2H),6.37(d,J＝8.9Hz,2H),3.31-3.27(m,12H),2.10(s,2H),1.17-1.12(m,24H)； 13 C NMR(101MHz,CDCl 3 )δ152.7,148.1,147.1,142.8,131.8,126.8,117.7,116.8,114.4,112.6,107.5,99.0,72.2,52.4,45.5,44.6,32.0,31.6,12.8,12.7.；ESI-MS(m/z):[M+H] + C 35 H 48 N 3 O 2 Calculated, 542.3747; found 542.3743.
Example 3: preparation of compound PHX3
107mg of the compound PHX3 was prepared as a magenta solid in 50% yield by a similar method to example 1.
1 H NMR(400MHz,CDCl 3 ):δ7.23(d,J＝8.7Hz,2H),6.93(d,J＝8.7Hz,1H),6.77(s,1H),6.74(d,J＝8.6Hz,1H),6.42(d,J＝7.7Hz,2H),6.36(dd,J＝8.8Hz,2.6Hz,2H),3.85-3.79(m,1H),3.37-3.27(m,8H),3.18(q,J＝7.1Hz,2H),2.09(s,2H),1.17-1.12(m,27H)； 13 C NMR(101MHz,CDCl 3 )δ152.7,148.1,131.5,126.8,117.5,116.9,107.5,99.0,72.3,53.6,52.4,44.6,32.0,31.6,20.2,12.7.；ESI-MS(m/z):[M+H] + C 36 H 50 N 3 O 2 Calculated, 556.3903; found 556.3901.
Example 4: preparation of compound PHX4
Prepared in a similar manner to example 1 to give 79mg of compound PHX4 as a purplish red solid in 37% yield.
1 H NMR(400MHz,CDCl 3 ):δ8.28(d,J＝2.7Hz,1H),8.09(dd,J＝9.0Hz,2.8Hz,1H),7.12-7.10(m,3H),6.46(d,J＝2.5Hz,2H),6.39(dd,J＝8.8Hz,2.5Hz,2H),3.35(q,J＝7.0Hz,8H),2.21(s,2H),1.26(s,6H),1.17(t,J＝7.0Hz,12H)； 13 C NMR(101MHz,CDCl 3 )δ161.0,152.7,148.5,141.6,132.4,126.2,123.7,123.7,118.0,114.6,107.4,98.8,51.2,44.5,31.9,31.8,12.6.；ESI-MS(m/z):[M+H] + C 31 H 38 N 3 O 4 Calculated, 516.2862; found 516.2864.
Example 5: preparation of compound PHX5
The compound 169mg of PHX5 was prepared as a white solid in 63% yield by a similar method to example 1.
1 H NMR(400MHz,CDCl 3 ):δ7.31(dd,J＝7.8Hz,1.6Hz,1H),7.21-7.16(m,3H),7.05(dd,J＝8.1Hz,1.2Hz,1H),6.98(td,J＝7.6Hz,1.3Hz,1H),6.43(d,J＝2.6Hz,2H),6.36(dd,J＝8.8Hz,2.6Hz,2H),3.37-3.28(m,8H),2.11(s,2H),1.17(s,6H),1.14(t,J＝7.0Hz,12H)； 13 C NMR(101MHz,CDCl 3 )δ155.1,152.7,148.2,131.5,127.5,127.0,126.6,120.7,117.4,116.4,107.5,99.0,72.9,52.2,44.6,32.1,31.3,12.7.；ESI-MS(m/z):[M+H] + C 31 H 39 N 2 O 2 Calculated, 471.3012; found 471.3010.
Example 6: preparation of compound PHX6
Prepared in a similar manner to example 1 to give 125mg of compound PHX6 as a white solid in 51% yield.
1 H NMR(400MHz,CDCl 3 ):δ7.18(d,J＝8.7Hz,2H),6.95(d,J＝8.8Hz,1H),6.85(d,J＝2.9Hz,1H),6.76(dd,J＝8.8Hz,2.9Hz,1H),6.42(d,J＝2.1Hz,2H),6.36(dd,J＝8.7Hz,2.1Hz,2H),3.82(s,3H),3.32(q,J＝7.0Hz,8H),2.13(s,2H),1.18(s,6H),1.14(t,J＝7.0Hz,12H)； 13 C NMR(101MHz,CDCl 3 )δ153.7,152.7,149.2,148.2,132.7,126.7,118.0,116.3,112.8,112.0,107.4,98.9,72.7,55.9,52.1,44.6,31.9,31.8,12.7；ESI-MS(m/z):[M+H] + C 32 H 41 N 2 O 3 Calculated, 501.3117; found 501.3115.
Example 7: preparation of compound PHX7
Similar procedure as in example 1 gave 118mg of compound PHX7 as red solid in 55% yield.
1 H NMR(400MHz,CDCl 3 ):δ7.47(d,J＝2.7Hz,1H),7.22(d,J＝8.9Hz,1H),6.78(dd,J＝9.0Hz,2.8Hz,1H),7.06(d,J＝8.8Hz,2H),6.43(d,J＝2.5Hz,2H),6.27(dd,J＝8.8Hz,2.5Hz,2H),3.36(q,J＝7.0Hz,8H),2.20(s,2H),1.25(s,6H),1.16(t,J＝7.0Hz,12H)； 13 C NMR(101MHz,CDCl 3 )δ160.1,153.5,147.1,140.6,131.2,125.3,122.8,121.8,119.0,113.6,106.0,98.8,51.3,44.5,32.2,32.1,12.5.；ESI-MS(m/z):[M+H] + C 31 H 37 BrN 2 O 2 Calculated, 548.2038; found 548.2036.
Example 8: preparation of compound PHX8
In a similar manner to example 1, 99mg of the compound PHX8 was obtained as a purplish red solid with a yield of 40%.
1H NMR(400MHz,CDCl3):δ7.82(d,J＝2.6Hz,1H),7.68(dd,J＝9.0Hz,2.7Hz,1H),7.08-7.06(m,3H),6.42(d,J＝2.6Hz,2H),6.36(dd,J＝8.8Hz,2.6Hz,2H),3.34(q,J＝7.1Hz,8H),2.22(s,2H),1.27(s,6H),1.16(t,J＝7.1Hz,12H)；13C NMR(101MHz,CDCl3)δ160.8,153.2,149.0,141.8,133.1,125.9,124.1,123.4,118.1,113.9,108.1,99.3,50.4,44.7,31.8,31.1,12.6.；ESI-MS(m/z):[M+H]+C 32 H 37 N 3 O 2 Calculated, 495.2886; found 495.2884.
Example 9: preparation of compound PHX9
In a similar manner to example 1, 122mg of the compound PHX9 was obtained as a pink solid in 58% yield.
1 H NMR(400MHz,CDCl 3 ):δ7.27(d,J＝8.7Hz,2H),6.97(dd,J＝7.4,1.9Hz,1H),6.77-6.69(m,2H),6.48(d,J＝2.5Hz,2H),6.43(dd,J＝8.7,2.5Hz,2H),2.93(s,12H),2.91(s,6H),2.07(s,2H),1.15(s,6H).13C NMR(101MHz,CDCl3)δ152.4,150.8,147.4,145.7,131.5,126.5,117.8,117.6,114.1,112.2,108.1,99.8,52.2,42.2,40.7,32.0,31.6.ESI-MS(m/z):[M+H] + C 29 H 35 N 3 O 2 Calculated, 457.2729; found 457.2727.
Example 10: preparation of compound PHX10
In a similar manner to example 1, 127mg of the compound PHX10 was obtained as a pink solid with a yield of 60%.
1H NMR(400MHz,CDCl3):δ7.20(d,J＝8.8Hz,2H),6.94(dd,J＝7.4,1.9Hz,1H),6.78-6.71(m,2H),6.50(d,J＝2.6Hz,2H),6.41(dd,J＝8.8,2.6Hz,2H),3.70(t,J＝7.1Hz,8H),2.92(s,6H),2.23(m,4H),2.08(s,2H),1.16(s,6H).13CNMR(101MHz,CDCl3)δ152.8,151.1,146.6,146.0,132.1,126.8,118.7,118.6,114.3,112.5,107.9,99.6,54.6,52.0,43.1,41.1,32.4,16.3.ESI-MS(m/z):[M+H]+C 31 H 35 N 3 O 2 Calculated, 481.2729; found 481.2728.
Example 11
PHX1, PHX2, PHX3, PHX4, PHX5 and PHX6 were prepared as 10mM DMSO stock solutions, respectively, and each stock solution was diluted 1000-fold with pure water to prepare 10. Mu.M DMSO probe aqueous solution containing 0.1%. Adjusting the pH value of the solution by using 0.01M/0.1M/1M/10M HCl and 0.01M/0.1M/1M/10M NaOH solution, wherein the pH adjustment interval is 0.1-0.25pH unit, and reading after the reading is stabilized for 2min after each adjustment. And measuring the corresponding ultraviolet visible absorption and fluorescence emission curves under each pH, reading the ultraviolet absorbance at 553nm to make a dot diagram of the pH, and performing nonlinear fitting, and reading the fluorescence emission intensity at 575nm to make a dot diagram of the pH and performing nonlinear fitting. Non-linear fitting of PHX1, PHX2, PHX3 the fitted curve was split into Hill-and HH-components using the Origin 8.0biphasic dose-response function using equation one. The non-linear fit of PHX4, PHX5 and PHX6 uses the Origin 8.0dose-response function.
Apparent pK of PHX1, PHX2, PHX3 read from UV and fluorescence titration curves a Respectively 6.21/5.88, 6.85/6.85 and 6.35/6.49, and the acid-base response range delta pH 10-90 Are all less than 1pH unit, and are respectively 0.9, 0.8 and 0.7pH units, and Hill-coefficient (Hill-coefficient) are respectively 4.1, 4.4 and 7.0. PHX4, PHX5 and PHX6 apparent pK read from UV and fluorescence titration curves a 5.63/5.84, 5.41/5.76 and 5.60/6.01 respectively, the Hill constant is between 1 and 1.6, and the acid-base response range is between 1.2 and 2pH units. The results are shown in FIGS. 1 to 6. These results show that the proton synergistic mechanism can realize the construction of Hill-type acid-base fluorescent probe, and the apparent pK can be realized by adjusting the substituent (dimethyl, diethyl and ethyl isopropyl) on the nitrogen atom a And acid-base response range Δ pH 10-90 And effective adjustment of the hill constant.
The results show that the compounds of the invention, especially PHX1, PHX2, PHX3, are typical Hill-type fluorescent probes, have a narrow acid-base response range, and can be used for detecting slight pH (0.1-0.2 pH) fluctuation in solution systems.
Example 12: cellular imaging
In vitro HeLa cell imaging of PHX2 and cell co-localization experiments. HeLa cells were incubated with a medium containing 0.5. Mu.M PHX2, 0.5. Mu.M rhodamine 123 (Invitrogen) or 0.5. Mu.M LysoSensor Green DND-189 (Invitrogen) for 30 minutes, washed three times with PBS to remove excess dye, and 1mL of the medium was added to maintain the morphology of the cells. Cells were imaged using an Olympus FV1000 confocal microscope with a 60x objective. FITC filter settings for each dye, PHX2: excitation is 559nm, and emission is 570-630nm; lysoSensor Green DND-189: excitation at 405nm and emission at 460-530nm; rhodamine 123: excitation at 488nm and emission at 500-550nm.
As can be seen from FIGS. 7 (a-c), an incubation time of 5 minutes was sufficient to allow PHX2 to cross the cell membrane to the interior of the cell, exhibiting a punctate staining pattern. Since PHX2 is pK a 6.9, PHX2 and the mitochondrial localization dye rhodamine 123 showed almost no overlap in staining site (FIG. 7,g-i), while PHX2 and the lysosomal localization dye LysoSensor Green DND-189 showed partial overlap in staining site (FIG. 7,d-f). PHX2 has no specific subcellular organelle localization effect, may be distributed on lysosomes, acidic subcellular organelles such as peroxidase or endocytosis and the like to a certain extent, does not show strong fluorescence brightness in mitochondria and cannot be excluded from being distributed in mitochondria.
The PHX series probe has strong cell permeability, and can stain cells for fluorescence imaging.
Example 13: cell level pH assay
The cell confocal imaging experiment under different pH values takes 5 multiplied by 10 of the logarithmic growth phase 4 Hepa1-6 cells, at a content of 5% CO 2 Was cultured overnight in a 37 ℃ incubator. Removing the culture medium, adding a medium containing 15. Mu.M PHX1, PHX2, PHX3 or 5. Mu.M
Medium of Red AM (purchased from Invitrogen) and incubated for 15 min. The medium was removed and a 5 μ g/mL solution of nigericin (nigericin, purchased from Thermo Fisher) in PBS (pH = 7.4) was added and incubated for 10 minutes. Before imaging, cells were washed three times with PBS (pH = 7.4) and then plates were plated with high concentrations of K at different pH values + The pH of the buffer solution is 7.1, 6.9,6.7, 6.5, 6.1,5.8,5.5,5.2 and 5.0 respectively. Imaging was performed using an Olympus fluorescence inverted microscope (x 20 objective). PHX1-3
The excitation wavelength of Red AM is 559nm, and the collection range of emission wavelength is 565-640nm.
In conclusion, PHX1-3 also has a narrower corresponding range of acid and base in a cell imaging system, and can give obvious signal response to small change of 0.2-0.3pH unit.
Example 14: cancer tissue differentiation
Will be 5X 10 6 Hepa1-6 cells were transplanted into eight-week-old male BALB/c nude mice intrahepatically to generate liver cancer tissues, and the liver cancer tissues were imaged 20 days later. Fresh sections, approximately 0.5-1mm thick, were removed from liver tumor and normal liver tissue with a scalpel and exposed directly to 15 μ M PHX2 in PBS for 15 minutes without fixation. Tissue sections were imaged with a confocal laser microscope (FV 1200, olympus,40 × water lens) with an excitation light of 559nm, emission collection band of 590-640nm, all image resolutions 1024 × 1024 pixels, and a zoom value of 2.0. Three regions of interest (ROI) were selected and their mean fluorescence intensity was calculated (FIG. 9,F)And G), wherein ROI1 is the nuclear region, ROI2 is the cytoplasmic region, and ROI3 is the extracellular region. Specimens of confocal imaging sites were fixed in 4% formalin buffer and embedded in paraffin, and sectioned for hematoxylin and eosin (H) for assessment of histopathological changes&E) And (6) dyeing. The sections were visualized and scanned using OlyVIA software 2.6 (Olympus, munster, germany).
PHX2 does not enter the nucleus (ROI 1), so the fluorescence intensity of ROI1 is weak in imaging of either tumor tissue sections or normal tissue sections. Because the pH range in the tumor tissue cells is 7.2-7.4, and the PHX2 fluorescence is weak in the range, the fluorescence intensity of cytoplasm ROI2 is low, while the acidic microenvironment (namely the tumor tissue intercellular substance) pH of the tumor cells is 6.5-6.9, the fluorescence of PHX2 can be enhanced, so that the fluorescence intensity of ROI3 is obviously higher than that of ROI1 and ROI2 (figure 9,F), and PHX2 can selectively lighten the tumor intercellular substance (extracellular region) to image the pH gradient reversed inside and outside the cells.
In contrast to the staining of normal liver tissue sections, the fluorescence of the nuclear ROI1, cytoplasmic ROI2 and extracellular ROI3 was overall weak (FIG. 9,G), but the fluorescence intensity of the intracellular ROI2 was relatively bright. This is because the pH in normal tissue cells is about 7.2, and the pH outside the cells is about 7.4, and therefore, in the case of PHX2 staining, the fluorescence intensity inside the cells is slightly higher than outside the cells, but the entire cells are in a substantially bright state. This contrasts strongly with the intracellular and extracellular fluorescence intensity of the cancer tissue section staining, and the diagnosis result is also consistent with the pathological section diagnosis result of the liver cancer tissue and the normal tissue (fig. 9,B and C).
PHX2 can identify the reverse pH gradient inside and outside cancer tissue cells, and has the potential of rapidly diagnosing solid tumor tissues.
Claims (13)
1. A compound of the following formula I:
formula I
In the formula, R 1 And R 2 Are each C1-C4 alkyl;
R 3 and R 4 Are each C1-C4 alkyl;
R 5 and R 6 Are each C1-C4 alkyl;
R 7 is NR a R b Wherein R is a And R b Are each C1-C4 alkyl.
2. The compound of claim 1, wherein the compound is selected from the group consisting of:
3. a compound represented by the following formula II:
in the formula (I), the compound is shown in the specification,
R 1 and R 2 Are each C1-C4 alkyl; and
R 3 and R 4 Are each C1-C4 alkyl.
4. A composition comprising a compound of claim 1 or 2.
5. The composition of claim 4, wherein the composition is a PBS solution or DMSO-containing aqueous solution of the compound of claim 1 or 2; or the composition is a cell culture medium containing the compound of claim 1 or 2.
6. A process for the preparation of a compound according to claim 1, comprising the step of reacting a compound of formula II according to claim 3 with a compound of formula III below in a solvent at 0 ℃ or below:
formula III
In the formula III, R 5 And R 6 Are each C1-C4 alkyl; r 7 Is NR a R b Wherein R is a And R b Are each C1-C4 alkyl.
7. The process according to claim 6, wherein the solvent is THF or Et 2 O, dioxane, or any mixture thereof.
8. The method of claim 6, wherein the reaction temperature is from 0 ℃ to-78 ℃.
9. The method of claim 6, wherein the reaction time is 1 to 5 hours.
10. The method of claim 6, wherein the molar ratio of the compound of formula II to the compound of formula III is between 1:1 and 1.5.
11. The method according to claim 6, wherein the reaction temperature is raised to room temperature after the completion of the reaction, and then the reaction is quenched by adding a saturated ammonium chloride solution.
12. A kit comprising a compound according to claim 1 or 2 or a composition according to claim 4 or 5.
13. Use of a compound of formula I according to claim 1 or 2 for the preparation of a reagent or kit for real-time monitoring of pH fluctuations or for cancer diagnosis.
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| CH553179A (en) * | 1969-12-23 | 1974-08-30 | Ciba Geigy Ag | Cryochromic diamino (thio) xantheno spiro dibenzo pyrans - prepd. from xanthylium salt and Grignard cpd. |
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