3-cyano phenalenone compound and application thereof
阅读说明：本技术 3-氰基非那烯酮类化合物及其应用 (3-cyano phenalenone compound and application thereof ) 是由 张志超 王紫千 于 2020-04-16 设计创作，主要内容包括：3-氰基非那烯酮类化合物及其应用,其属于荧光标记的技术领域。本发明采用多种手段检测了3-氰基非那烯酮类化合物与PKM2的结合能力,该类化合物可以有效的共价结合PKM2蛋白。并通过凝胶荧光成像、活细胞荧光成像实验检测了它们在体外和活细胞(包括肿瘤细胞和正常组织细胞)内特异性荧光标记PKM2蛋白的能力。表明该类化合物可以有效的在体外和活细胞内与PKM2发生亲核取代反应,并生成具有橙红色荧光的结构,实现对于PKM2的特异性荧光标记。肿瘤细胞和正常细胞的活细胞荧光成像实验进一步表明该类化合物能够特异性的对肿瘤细胞进行荧光染色,而不对正常细胞进行荧光染色,实现对于肿瘤细胞、组织的荧光可视化和肿瘤细胞、组织的分子影像学诊断。(3-cyano phenalenone compounds and application thereof, which belong to the technical field of fluorescent labeling. The invention adopts a plurality of means to detect the binding capacity of the 3-cyano phenalenone compound and the PKM2, and the compound can be effectively and covalently bound with the PKM2 protein. And the gel fluorescence imaging and living cell fluorescence imaging experiments are used for detecting the capability of the protein of PKM2 of specific fluorescence labeling in vitro and in living cells (including tumor cells and normal tissue cells). The compounds can effectively perform nucleophilic substitution reaction with PKM2 in vitro and in living cells, generate an orange-red fluorescent structure, and realize specific fluorescent labeling on PKM 2. The living cell fluorescence imaging experiment of the tumor cells and the normal cells further shows that the compounds can specifically carry out fluorescence staining on the tumor cells, but not on the normal cells, thereby realizing the fluorescence visualization of the tumor cells and tissues and the molecular imaging diagnosis of the tumor cells and tissues.)
A 3-cyanophenanonene compound having the general formula I:
wherein:
R1is selected from C1-4Alkenyl radical, C1-4Alkynyl radical, F, C2F5、CF3、NO2、CN、CHO、COOH、SO3H、COR3、COOR3、CONHR3、SO3R3、SO2NHR3；
R2Selected from the group consisting of piperidinyl,Piperidonyl, piperiddionyl, pyrrolidinyl, pyrrolidonyl, pyrrolindionyl or-XR4；
R3Selected from substituted or unsubstituted C1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl, said substitution being optionally substituted with: OH, I, Br, Cl, F, NO2、NHCH3、N(CH3)2、CN、CF3；
R4Selected from the group consisting of substituted or unsubstituted phenyl, naphthyl, thienyl, furyl, pyrrolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, pyrimidinyl, indolyl, benzothienyl, benzindolyl, benzofuryl, quinolinyl, isoquinolinyl, or purinyl, wherein said substitution is optionally substituted with: c1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl, phenyl, OH, I, Br, Cl, F, NO2、NHCH3、N(CH3)2、CN、CF3、CHO、COOH、SO3H、COR5、COOR5、CONHR5、SO3R5、SO2NHR5OR OR5；
R5Is selected from C1-4Alkyl radical, C1-4Alkenyl radical, C1-4An alkynyl group;
x is selected from S or O.
2. The 3-cyanophenanones according to claim 1, wherein R is selected from the group consisting of1Selected from CN and COR3、COOR3。
3. The 3-cyanophenanones according to claim 2, wherein R is selected from the group consisting of3Is selected from C1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl.
4. The 3-cyanophenanones according to claim 1, wherein R is selected from the group consisting of2Is selected from-XR4。
5. The 3-cyanophenanones according to claim 4, wherein R is selected from the group consisting of4Selected from phenyl, 2-methylphenyl, 3-methylphenyl, 4-ethylphenyl, 3-isopropylphenyl, 4-tert-butylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-methoxyphenyl and 4-dimethylaminophenyl.
6. The 3-cyanophenanones according to claim 4, wherein X is S.
7. The 3-cyanophenanones according to claim 1, wherein: 3-cyanophenanones selected from:
8. the use of a 3-cyanophenanonene compound according to claim 1 for the preparation of a functional reagent for the fluorescent labeling of PKM2 protein.
9. The use of the 3-cyanophenanonenes according to claim 1 for the preparation of functional reagents for the detection of biological information such as the expression level, spatio-temporal distribution of PKM2 family proteins.
10. The use of the derivatives of 3-cyanophenanonenes according to claim 1 for the preparation of functional reagents for the fluorescence visualization of tumor cells and the molecular imaging diagnosis of tumor cells and tissues.
Technical Field
The invention relates to 3-cyano phenalenone compounds, in particular to 6-benzene sulfenyl-3-cyano phenalenone and derivatives thereof. In particular to the application of the compounds in vitro, in vivo and in vivo for specific fluorescence labeling of M2 type pyruvate kinase (PKM 2); and the application of the compounds as molecular diagnostic probes for realizing tumor cell visualization.
Background
Molecular imaging, which is the qualitative and quantitative research on living body, tissue, cell and even molecular level by applying imaging related method to biological process in living body state, has wide application in early stage image diagnosis of tumor, cell signal, disease mechanism research, drug research and development, drug delivery, drug effect evaluation, etc. The fluorescence analysis method has the advantages of high sensitivity, good selectivity, wide dynamic response range, measurement conditions closer to the physiological environment of a living body and the like, and is widely applied to molecular imaging analysis. The chemical small molecule probe is used for specific fluorescent labeling of protein highly related to occurrence, development and treatment of specific diseases, namely molecular markers of the diseases, so that the fluorescence visualization of the content, activity and tissue distribution of tumor marker protein can be realized on the level of cells, tissues and living bodies, the tissue occurrence, cell differentiation and cell functions of the related diseases can be known, and the diagnosis, classification, prognosis judgment and treatment guidance of tumors are realized.
Muscle-type Pyruvate Kinase (PKM) is a key enzyme that regulates the last step of glycolysis, catalyzing the reactions of phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP. PKM is divided into two subtypes, pyruvate kinase M1 (PKM1) and pyruvate kinase M2 (PKM 2). The expression levels of PKM1 and PKM2 in different cells vary greatly: PKM1 is expressed primarily in the muscle and brain, differentiated end-cells that have a vigorous demand for energy; the PKM2 is mainly expressed in cells with strong anabolic requirements such as embryonic cells, stem cells and tumor cells, and plays a crucial role in the growth process of tumor cells by regulating Waeberg. Therefore, the PKM2 is a molecular marker of various tumor cells, namely the content of PKM2 in the cells and tissues can indicate whether the cells and tissues have the properties of tumors. By detecting the content of PKM2 in cells and tissues, the histogenesis, cell differentiation and cell function of tumors can be known, and the diagnosis, classification, prognosis judgment and treatment guidance of the tumors are realized.
Therefore, the small molecular fluorescent labeling probe aiming at the PKM2 protein is designed and developed, can be applied to specific fluorescent labeling and fluorescent visualization of a tumor molecular marker PKM2 protein on the level of cells, tissues and living bodies, can realize the fluorescent visualization detection of tumor cells and the molecular imaging diagnosis, classification and prognosis judgment of the tumor cells and tissues, can guide the treatment of tumors, and has important significance for realizing accurate diagnosis and treatment of the tumors.
Disclosure of Invention
The present invention aims to obtain the following molecules: specific fluorescent labeling of small molecule fluorescent probes for pyruvate kinase M2 (PKM2) in vitro, in vivo and in vivo; functional molecules for detecting biological information such as expression level, spatial-temporal distribution and the like of PKM2 family proteins; functional molecules for carrying out fluorescence visualization detection and tumor molecular imaging diagnosis on tumor cells.
In one aspect, the invention provides a 3-cyanophenanonene compound having the structure of formula I:
wherein:
R1is selected from C1-4Alkenyl radical, C1-4Alkynyl radical, F, C2F5、CF3、NO2、CN、CHO、COOH、SO3H、COR3、COOR3、CONHR3、SO3R3、SO2NHR3；
R2Selected from piperidyl, piperidonyl, piperiddionyl, pyrrolidinyl, pyrrolidonyl, pyrrolindionyl or-XR4；
R3Selected from substituted or unsubstituted C1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl, said substitution beingOptionally substituted with: OH, I, Br, Cl, F, NO2、NHCH3、N(CH3)2、CN、CF3；
R4Selected from the group consisting of substituted or unsubstituted phenyl, naphthyl, thienyl, furyl, pyrrolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, pyrimidinyl, indolyl, benzothienyl, benzindolyl, benzofuryl, quinolinyl, isoquinolinyl, or purinyl, wherein said substitution is optionally substituted with: c1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl, phenyl, OH, I, Br, Cl, F, NO2、NHCH3、N(CH3)2、CN、CF3、CHO、COOH、SO3H、COR5、COOR5、CONHR5、SO3R5、SO2NHR5OR OR5；
R5Is selected from C1-4Alkyl radical, C1-4Alkenyl radical, C1-4An alkynyl group;
x is selected from S or O.
R1Preferably CN and COR3、COOR3。
R2Preferred XR4。R4Preferred are phenyl, 2-methylphenyl, 3-methylphenyl, 4-ethylphenyl, 3-isopropylphenyl, 4-tert-butylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-methoxyphenyl and 4-dimethylaminophenyl.
The R is3Preferably selected from C1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl.
X is preferably S.
In a more specific technical scheme, the sulfur/oxygen phenalene compound is selected from compounds with the following structures:
for the above-mentioned cyanophenanones, the present invention examined their ability to bind to PKM2 by various means. The results show that the compound of the invention with the general formula I can be effectively and covalently bound to the PKM2 protein. The invention tests the capability of the protein of PKM2 to be specifically and fluorescently labeled in vitro and in living cells (including tumor cells and normal tissue cells) through gel fluorescence imaging and living cell fluorescence imaging experiments. The results show that the compounds can effectively perform nucleophilic substitution reaction with PKM2 in vitro and in living cells, generate a structure with orange red fluorescence, and realize specific fluorescence labeling on PKM 2. The living cell fluorescence imaging experiment of the tumor cells and the normal cells further shows that the compounds can specifically carry out fluorescence staining on the tumor cells, but not on the normal cells, thereby realizing the fluorescence visualization of the tumor cells and tissues and the molecular imaging diagnosis of the tumor cells and tissues.
Drawings
Figure 1 is a plot of the change in fluorescence of compounds 5 and 9 upon in vitro co-incubation with PKM2 protein.
FIG. 2 shows the results of gel fluorescence experiments of
FIG. 3 is a graph showing the confocal effect of Compound 9 in Hela cells. The green channel is a labeling effect graph of the probe molecules, and the red channel is an effect graph of labeling the PKM2 protein by an immunofluorescence method.
FIG. 4 shows the result of fluorescent staining of MCF-7, Hela and HEK-293T cells, which are tumor cells, with Compound 9 under a fluorescent microscope.
Detailed Description
The 3-cyano phenalenone compound provided by the invention has a structure shown in a general formula I
Wherein: r1Is selected from C1-4Alkenyl radical, C1-4Alkynyl radical, F, C2F5、CF3、NO2、CN、CHO、COOH、SO3H、COR3、COOR3、CONHR3、SO3R3、SO2NHR3；
R2Selected from piperidyl, piperidonyl, piperiddionyl, pyrrolidinyl, pyrrolidonyl, pyrrolindionyl or XR4；
R3Selected from substituted or unsubstituted C1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl, said substitution being optionally substituted with: OH, I, Br, Cl, F, NO2、NHCH3、N(CH3)2、CN、CF3；
R4Selected from the group consisting of substituted or unsubstituted phenyl, naphthyl, thienyl, furyl, pyrrolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, pyrimidinyl, indolyl, benzothienyl, benzindolyl, benzofuryl, quinolinyl, isoquinolinyl, or purinyl, wherein said substitution is optionally substituted with: c1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl, phenyl, OH, I, Br, Cl, F, NO2、NHCH3、N(CH3)2、CN、CF3、CHO、COOH、SO3H、COR5、COOR5、CONHR5、SO3R5、SO2NHR5OR OR5；
R5Is selected from C1-4Alkyl radical, C1-4Alkenyl radical, C1-4An alkynyl group;
x is selected from S or O.
R1Preferably CN and COR3、COOR3。
R2Preferred XR4。R4Preferred are phenyl, 2-methylphenyl, 3-methylphenyl, 4-ethylphenyl, 3-isopropylphenyl, 4-tert-butylphenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl, 4-methoxyphenyl and 4-dimethylaminophenyl.
Said R3Is selected from C1-4Alkyl radical, C1-4Alkenyl radical, C1-4Alkynyl.
X is preferably S.
In a more specific technical scheme, the sulfur/oxo phenalenone compound is selected from compounds with the following structures:
the present invention further provides a method for preparing the 3-cyanophenanones of the present invention, comprising the steps of:
(1) 5-bromoacenaphthenequinone and R2CH2CN reacts according to the feeding molar ratio of 1:1-1:1.5 to prepare a compound II; the reaction time is 3-12h, the reaction temperature is 60-120 ℃, and the reaction solvent is acetonitrile;
(2) heating the compound II in the presence of an acid-binding agent for reaction to obtain a compound III, wherein the reaction time is 3-12h, the reaction temperature is 60-120 ℃, the reaction solvent is acetonitrile, and the acid-binding agent is potassium carbonate;
(3) compounds III and R1XH is added into the acid binding agent according to the molar ratio of 1: 1.2-1: 5, reacting to obtain a compound I, wherein the reaction time is 3-12h, the reaction temperature is 60-150 ℃, the reaction solvent is acetonitrile, and the acid-binding agent is potassium carbonate; this step is preferably carried out under nitrogen protection;
in all the embodiments of the present invention mentioned above, the term "alkyl" used includes straight chain alkyl and branched chain alkyl. Reference to a single alkyl group, such as "propyl", is intended to refer only to straight chain alkyl groups, and reference to a single branched alkyl group, such as "isopropyl", is intended to refer only to branched alkyl groups. Also as "C1-4Alkyl "includes C1-3Alkyl, methyl, ethyl, n-propyl, isopropyl and tert-butyl. Similar rules apply in this specificationOther groups used.
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and the raw materials and reagents used were all purchased from chemical or biological reagents companies or prepared by published methods.