CN115043833A - Bcl-2 fluorescent probe and preparation method and application thereof - Google Patents
Bcl-2 fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN115043833A CN115043833A CN202110254474.9A CN202110254474A CN115043833A CN 115043833 A CN115043833 A CN 115043833A CN 202110254474 A CN202110254474 A CN 202110254474A CN 115043833 A CN115043833 A CN 115043833A
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- bcl
- protein
- fluorescent probe
- compound
- molecule fluorescent
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Abstract
The invention relates to a Bcl-2 small-molecule fluorescent probe and a preparation method and application thereof, and the structure of the compound is shown as a general formula I, wherein R is 1 、R 2 、R 3 R and X are as defined in the specification. The probe has novel structure, strong binding affinity to Bcl-2 protein, high selectivity and good fluorescence characteristic. The compound can be used for preparing a high-throughput screening reagent of a Bcl-2 protein inhibitor, an early diagnosis of cancer and a marking and sorting reagent of tumor cells.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a Bcl-2 small-molecule fluorescent probe, a preparation method thereof and application thereof in Bcl-2 protein specific quantitative detection, Bcl-2 inhibitor screening, cancer detection and cell sorting.
Background
The Bcl-2 family proteins as an important signal molecule can be divided into pro-apoptotic proteins (Bax, Bak and the like), anti-apoptotic proteins (Bcl-2,Mcl-1，Bcl-X L etc.) and BH3-only protein (Bad, Bid, Bim and Noxa, etc.). The complex interaction between the Bcl-2 family pro-apoptotic protein and the anti-apoptotic protein can accurately regulate and control the endogenous apoptotic pathway mediated by mitochondria, and has important significance for the development of multicellular organisms and the maintenance of the homeostasis of the organisms.
Under normal physiological conditions, the expression quantity of the anti-apoptosis protein and the pro-apoptosis protein of the Bcl-2 family maintains dynamic balance so as to realize accurate regulation and control of an endogenous apoptosis pathway, but certain pathological stimulation or gene mutation can cause the expression imbalance of the anti-apoptosis protein and the pro-apoptosis protein, so that a series of diseases such as autoimmune diseases, neurodegenerative diseases, cancers and the like are caused. In addition, over-expression of anti-apoptotic proteins antagonizes the function of pro-apoptotic proteins, inhibits the apoptotic process, and prevents the body from clearing abnormal or diseased cells. Research shows that overexpression of Bcl-2 family anti-apoptosis proteins (such as Bcl-2, Mcl-1 and other proteins) is a main reason for apoptosis escape of tumor cells, and overexpression of the Bcl-2 family anti-apoptosis proteins becomes one of markers of the tumor cells, and the overexpression of the Bcl-2 family anti-apoptosis proteins are found in various tumor cells such as breast cancer, lung cancer, prostatic cancer, ovarian cancer, pancreatic cancer, cervical cancer, leukemia, melanoma and the like. Therefore, we can distinguish tumor cells from normal cells by detecting the expression amount of the anti-apoptotic protein in the cells.
At present, several Bcl-2 protein fluorescent probes are developed, but the application of the fluorescent probes is greatly limited due to the problems of low affinity and poor selectivity of the fluorescent probes to target proteins. In addition, the existing Bcl-2 fluorescent probe cannot detect the expression level of the Bcl-2 protein in living cells. In order to solve the problems of the existing Bcl-2 protein fluorescent probes, a series of novel Bcl-2 protein fluorescent probes are developed, show extremely high affinity and selectivity for Bcl-2 protein, and are successfully applied to the detection of the Bcl-2 protein expression quantity in living cells.
Disclosure of Invention
The invention provides a Bcl-2 small-molecule fluorescent probe, a preparation method thereof and application thereof in preparation of Bcl-2 protein quantitative determination, cancer early diagnosis, a tumor cell labeling reagent and a sorting reagent.
The specific technical scheme is as follows:
first, Bcl-2 small molecule fluorescent probe
A Bcl-2 small-molecule fluorescent probe has a structure shown in the following structural general formula I:
wherein R is 1 Represents a hydrogen atom, a halogen atom, C 1-6 Alkyl or C 1-6 A halogenated alkyl group,
R 2 、R 3 each independently represents a hydrogen atom, a halogen atom, C 1-6 Alkyl or C 1-6 A halogenated alkyl group,
R 4 represents a hydrogen atom, C 1-6 Alkyl or C 3-8 A cycloalkyl group, which is a cyclic alkyl group,
x is N or CH ═ CH,
r is a fluorescent group.
Preferably, the first and second electrodes are formed of a metal,
said R is 1 Represents a hydrogen atom, a fluorine atom, a chlorine atom, C 1-4 Alkyl or C 1-4 A halogenated alkyl group,
R 2 、R 3 each independently represents a hydrogen atom, a halogen atom, C 1-4 Alkyl or C 1-4 A halogenated alkyl group,
x is CH (CH-CH),
R 4 represents a hydrogen atom, C 1-4 Alkyl or C 3-6 A cycloalkyl group,
r is 5-dimethylamino-1-sulfonyl naphthalene, 8-aminoquinoline, 8-hydroxyquinoline, 7-amino-4-methylcoumarin, 7-hydroxy-4-methylcoumarin, 8-sulfonylamino quinoline, 2-sulfonylamino-10-ethyl acridone, 4- (N-benzopyrrolinone) -benzene sulfonamide and tetraphenyl ethylene derivatives.
It is further preferred that the first and second liquid crystal compositions,
the Bcl-2 small molecule fluorescent probe is selected from compounds with the following structural formula:
preparation method of Bcl-2 small-molecule fluorescent probe
A preparation method of a Bcl-2 small-molecule fluorescent probe comprises the following steps:
3, 3-dimethylcyclohexanone reacts with phosphorus oxychloride and N, N-dimethylformamide to generate an intermediate 2; the intermediate 2 reacts with p-chlorobenzeneboronic acid to generate an intermediate 3; 4-chloro-2-fluorobenzoic acid is protected by methyl ester to obtain an intermediate 4, then the intermediate 4 is reacted with Boc-piperazine to generate an intermediate 6, then an intermediate 7 is obtained by nucleophilic substitution reaction, and an intermediate 8 is obtained after the Boc protecting group is removed; then the intermediate 8 is subjected to reductive amination reaction to obtain an intermediate 9, and the intermediate 10 is obtained after hydrolysis; reacting the intermediate 10 with fluorophores with different structures to obtain a compound with a general formula I;
the reaction route is as follows:
wherein R, R1, R2, R3 and R4 are as defined in formula I above;
reaction reagents and reaction conditions: (a) phosphorus oxychloride, N-dimethylformamide, dichloromethane; (b) [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride, substituted aryl boric acid or heterocyclic boric acid, 1, 4-dioxane and water; (c) refluxing acetyl chloride and methanol; (d) palladium acetate, cesium carbonate, toluene, 80 ℃; (e) sodium hydride, N-dimethylformamide, 140 ℃; (f) hydrogen chloride saturated ethyl acetate, dichloromethane; (g)3, 1, 2-dichloroethane, sodium triacetoxyborohydride; (h) LiOH, tetrahydrofuran/water; (i) R-NH 2 Isobutyl chloroformate, N-methylmorpholine, tetrahydrofuran; or R-NH 2 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane.
Application of Bcl-2 small-molecule fluorescent probe
The invention relates to an application of a Bcl-2 small-molecule fluorescent probe in preparing a reagent for specifically identifying and quantitatively determining Bcl-2 protein;
the Bcl-2 small-molecule fluorescent probe is applied to the preparation of a reagent for realizing high-throughput screening of a Bcl-2 protein inhibitor and the preparation of a reagent for detecting and inhibiting the Bcl-2 protein;
the Bcl-2 small molecular fluorescent probe is applied to preparing a labeling and sorting reagent for tumor cells with high Bcl-2 protein expression;
the invention discloses an application of a Bcl-2 small-molecule fluorescent probe in preparing a reagent for early diagnosis of Bcl-2 protein overexpression related malignant tumors.
The Bcl-2 protein overexpression related malignant tumors in the application of the Bcl-2 small-molecule fluorescent probe are pancreatic cancer, lung cancer, prostatic cancer, breast cancer, ovarian cancer, cervical cancer, multiple myeloma and leukemia.
The "halogen" as referred to herein means a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like. Fluorine atom and chlorine atom are preferred.
The term "halo" as used herein means that any atom in the group which can be substituted is substituted by halogen, and can be perhalogenated, i.e., the halogen atom is substituted at all positions in the group which can be substituted.
Said "C" of the present invention 1-6 Alkyl "means a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 1, 2-dimethylpropyl, and the like. Preferably C 1-4 An alkyl group. Said "C" of the present invention 1-4 Alkyl "refers to the above examples containing 1 to 4 carbon atoms.
The 3-to 8-membered cycloalkyl group of the present invention includes a 3-to 8-membered saturated monocyclic cycloalkyl group and a 3-to 8-membered partially saturated monocyclic cycloalkyl group. 3-8 membered saturated monocyclic cycloalkyl, meaning that the monocyclic ring is fully saturated carbocyclic, examples of which include but are not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane, methylcyclohexane, dimethylcyclohexane, etc. 3-8 membered partially saturated monocyclic cycloalkyl, meaning that the monocyclic ring is a partially saturated carbocyclic ring, examples of which include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1, 4-cyclohexadienyl, cycloheptenyl, 1, 4-cycloheptadienyl, cyclooctenyl, 1, 5-cyclooctadienyl, and the like;
the term "C 3-8 Cycloalkyl group "," C 3-6 Cycloalkyl "is a specific example containing 3 to 8 and 3 to 6 carbon atoms in the following examples, respectively.
The invention has the beneficial effects that:
1. the probe of the invention has novel structure, extremely high affinity and selectivity to Bcl-2 protein, and the characteristics of trace, high efficiency and high specificity of the fluorescent probe.
2. The Bcl-2 fluorescent probe can selectively mark tumor cells, and can realize early diagnosis of cancers and related diseases and sorting of the tumor cells. The invention relates to the application of a Bcl-2 small-molecule fluorescent probe in the preparation of pharmaceutical preparations for screening Bcl-2 protein-related malignant tumors, wherein the related malignant tumors are malignant tumors; preferably pancreatic cancer, lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, multiple myeloma and leukemia.
Drawings
FIG. 1 shows (A) an absorption spectrum of Compound 11 (B) fluorescence excitation spectrum of the probe in PBS buffer solution (C) fluorescence emission spectrum of the probe in PBS buffer solution (D) fluorescence emission spectrum of the probe in different solvents (E) fluorescence emission spectrum of the probe in different solvents at different concentrations.
FIG. 2 shows (A) an absorption spectrum of Compound 12 (B) fluorescence excitation spectrum of the probe in PBS buffer solution (C) fluorescence emission spectrum of the probe in PBS buffer solution (D) fluorescence emission spectrum of the probe in different solvents (E) fluorescence emission spectrum of the probe in different solvents at different concentrations.
FIG. 3 shows (A) an absorption spectrum of Compound 13 (B) fluorescence excitation spectrum of the probe in PBS buffer solution (C) fluorescence emission spectrum of the probe in PBS buffer solution (D) at different concentrations (E) fluorescence emission spectrum of the probe in different solvents.
FIG. 4 shows (A) an absorption spectrum (B) of Compound 14, fluorescence excitation spectrum (C) of probe in PBS buffer solution at different concentrations, fluorescence emission spectrum (D) of probe in PBS buffer solution at different concentrations, fluorescence excitation spectrum (E) of probe in different solvents, and fluorescence emission spectrum of probe in different solvents.
FIG. 5 shows fluorescence properties of Compound 11 incubated with Bcl-2 protein. (A) Fluorescence emission spectra after incubation of 2 μ M of compound 11 with different concentrations of Bcl-2 protein (0.0125, 0.025,0.05,0.075,0.1 and 0.125mg/mL) for 20min at room temperature; (B) fluorescence emission spectra after incubation for 20min at room temperature with 2. mu.M Compound 11, 2. mu.M Compound 11+0.125mg/mL Bcl-2 protein + 5. mu.M ABT-199(Bcl-2 protein inhibitor); (C) fluorescence emission spectra of 2. mu.M Compound 11, 0.125mg/mL Bcl-2 protein, 2. mu.M Compound 11+0.125mg/mL Mcl-1 protein incubated in buffer at room temperature; (D) fluorescence intensity histograms after 20min incubation at room temperature with 2. mu.M Compound 11, 0.125mg/mL Bcl-2 protein, 2. mu.M Compound 11+0.125mg/mL Mcl-1 protein.
FIG. 6 shows fluorescence analysis of Compound 12 after incubation with Bcl-2 protein. (A) Fluorescence emission spectra after incubation of 1 μ M of compound 12 with different concentrations of Bcl-2 protein (0.025, 0.05,0.1,0.2 and 0.4mg/mL) in buffer solution for 20min at room temperature; (B) fluorescence emission spectra of 1. mu.M Compound 12, 1. mu.M Compound 12+0.4mg/mL Bcl-2 protein + 5. mu.M ABT-199(Bcl-2 protein inhibitor) incubated in buffer solution at room temperature; (C) fluorescence emission spectra after incubation for 20min at room temperature for 1. mu.M Compound 12, 1. mu.M Compound 12+0.4mg/mL Bcl-2 protein, 1. mu.M Compound 12+0.4mg/mL Mcl-1 protein, 1. mu.M Compound 12+0.4mg/mL BSA. (D) Histogram of fluorescence intensity after incubation for 20min at room temperature for 1. mu.M Compound 12, 1. mu.M Compound 12+0.4mg/mL Bcl-2 protein, 1. mu.M Compound 12+0.4mg/mL Mcl-1 protein, 1. mu.M Compound 12+0.4mg/mL BSA.
FIG. 7 shows the magnification of the lens for imaging after 1. mu.M Compound 11 was incubated with ACHN and HUVEC cells, respectively (A1: ACHN cells, brightfield imaging; A2: ACHN cells, fluorescence imaging; B1: HUVEC cells, brightfield imaging; B2: HUVEC cells fluorescence imaging) 63 ×.
FIG. 8(A) is the flow analysis of ACHN and HUVEC mixed cells incubated with 1 μ M Compound 11 for 20min at room temperature; (B) flow analysis of HL-60 and HUVEC mixed cells incubated with 1. mu.M Compound 11 for 20min at room temperature.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example preparation of 12-chloro-4, 4-dimethylcyclohex-1-en-1-carbaldehyde:
n, N-dimethylformamide (1.16mL, 15mmol) was dissolved in dichloromethane (30mL) and POCl was added under ice-bath 3 (1.05mL, 11.25mmol) and reacted at room temperature for 2 h. Then, 3-dimethylcyclohexanone (1.04mL, 7.5mmol) was added and reacted at room temperature for 4 h. The reaction solution was poured into ice water (120mL) and extracted 3 times with dichloromethane. The organic phases were combined, MgSO 4 Drying, spin-drying to obtain a crude product, and purifying by column chromatography to obtain a colorless oily liquid, wherein the yield is as follows: 76 percent. 1 H NMR(400MHz,CDCl 3 )δ10.22(S,1H),2.37(s,2H),2.32(t,J＝6.4Hz,2H),1.43(t,J＝6.4Hz,2H),0.98(s,6H).
Preparation of 4-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1, 1-biphenyl ] -2-carbaldehyde:
into a two-necked flask was added 4-chlorophenylboronic acid (0.75g, 4.81mmol), Pd (dppf) Cl 2 (0.11g, 0.148 mmol). Adding a solution of 2-chloro-4, 4-dimethylcyclohex-1-ene-1-carbaldehyde (0.64g, 3.7mmol) in 1, 4-dioxane and K 2 CO 3 (1.33g, 9.62mmol) was replaced with nitrogen three times, and then reacted at 85 ℃ for 6 hours. The heating was stopped, cooled to room temperature and 20mL of H was added to the mixture 2 O, and extracted three times with ethyl acetate. Filtering with a Celite pad to remove Pd (dppf) Cl 2 And concentrating the organic phase to obtain a crude product. Column chromatography gave a colorless oil, yield: 78 percent. 1 H NMR(400MHz,CDCl 3 )δ9.51(d,J＝11.1Hz,1H),7.35(d,J＝8.3Hz,2H),7.14(d,J＝8.4Hz,2H),2.38(dd,J＝7.5,5.5Hz,2H),2.28(s,2H),1.49(t,J＝6.5Hz,2H),1.01(s,6H).
Preparation of methyl 4-bromo-2-fluorobenzoate:
acetyl chloride (5.4mL, 75mmol) was slowly added to dry methanol (140mL) under ice-bath and reacted for 30 min. 4-bromo-2-fluorobenzoic acid (7.01g, 30mmol) was added to the solution and refluxed for 6 hours. The solvent was evaporated off and the crude product was washed with n-hexane to give a white solid, yield: 98% and a melting point of 58-60 ℃. 1 H NMR(400MHz,CDCl 3 )δ7.82(t,J＝8.2Hz,1H),7.35(t,J＝7.7Hz,2H),3.93(s,3H).
Preparation of tert-butyl 4- (3-fluoro-4- (methoxycarbonyl) phenyl) piperazine-1-carboxylate:
methyl 4-bromo-2-fluorobenzoate (0.47g, 2mmol), 1-Boc-piperazine (0.52g, 2.8mmol), Pd (OAc) 2 (0.0224g, 0.1mol) and BINAP (0.125g, 0.2mol), Cs 2 CO 3 (0.717g, 2.2mol) was charged into a two-necked flask, and dried toluene (20mL) was added to replace nitrogen, followed by reaction at 80 ℃ for 8 hours. The heating was stopped, cooled to room temperature and filtered through a pad of celite. The filtrate was spin-dried. The crude product was recrystallized from ethyl acetate to give a white solid, yield: 81%, melting point 148-. 1 H NMR(400MHz,CDCl 3 )δ7.84(t,J＝8.8Hz,1H),6.62(dd,J＝8.9,2.4Hz,1H),6.51(dd,J＝14.5,2.3Hz,1H),3.88(s,3H),3.68–3.50(m,4H),3.40–3.17(m,4H),1.49(s,9H).
Preparation of tert-butyl 4- (3- ((1H-pyrrolo [2,3-b ] pyridin-5-yl ] oxy) -4- (methoxycarbonyl) phenyl) piperazine-1-carboxylate:
dissolving tert-butyl 4- (3-fluoro-4- (methoxycarbonyl) phenyl) piperazine-1-carboxylate in anhydrous N, N-dimethylformamide (120mL), and adding 1H-pyrrolo [2,3-b]Pyridin-5-ol (2.1g, 15mmol) and NaH (0.66g, 16.5mmol) were replaced three times with nitrogen. The reaction was heated at 120 ℃ for 8 h. After the heating was stopped and the reaction mixture was cooled to room temperature, the reaction mixture was poured into water (300 mL). Extracted three times with ethyl acetate (150ml) and dried over anhydrous magnesium sulfate. The solvent is evaporated off, and the crude product is purified by column chromatography to obtain a white solid, wherein the yield is as follows: 76%, melting point 193-. 1 H NMR(400MHz,DMSO-d 6 )δ11.64(s,1H),8.01(d,J＝2.5Hz,1H),7.79(d,J＝8.9Hz,1H),7.48(t,J＝2.8Hz,1H),7.43(d,J＝2.5Hz,1H),6.79(dd,J＝9.0,2.1Hz,1H),6.43(d,J＝2.0Hz,1H),6.38(dd,J＝2.9,1.8Hz,1H),3.65(s,3H),3.42(t,J＝4.6Hz,4H),3.21(t,J＝4.7Hz,4H),1.39(s,9H).
Preparation of methyl 2- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy ] -4- (piperazin-1-yl) benzoate:
4- (3- ((1H-pyrrolo [2, 3-b)]Pyridin-5-yl]Tert-butyl oxy) -4- (methoxycarbonyl) phenyl) piperazine-1-carboxylate (4.5g, 10mmol) was dissolved in ethyl acetate (60mL) and HCl/EA (40mL) was added. After stirring overnight, it was filtered. To the filter cake was added ethyl acetate (100mL) and saturated NaHCO 3 (100mL) of the mixed solution was stirred at room temperature for 1 hour, and extracted three times with ethyl acetate. The organic phases were combined, anhydrous MgSO 4 Drying and spin-drying the solvent to obtain a white solid with yield: 96%, melting point 146-. 1 H NMR(400MHz,DMSO-d 6 )δ11.65(s,1H),8.01(d,J＝2.5Hz,1H),7.77(d,J＝9.0Hz,1H),7.48(t,J＝2.8Hz,1H),7.44(d,J＝2.5Hz,1H),6.78(dd,J＝9.0,2.1Hz,1H),6.46–6.29(m,2H),3.66(s,3H),3.16–3.05(m,4H),2.81–2.62(m,4H).
Preparation of methyl 2- ((1H-pyrrolo [2,3-b ] pyridinyl-5-yl) oxy ] -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoate:
2- ((1H-pyrrole [2, 3-b)]Pyridin-5-yl) oxy]Methyl (4- (piperazin-1-yl) benzoate (3.17g, 9mmol) was dissolved in 1, 2-dichloroethane (80mL), and 4-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1, 1-biphenyl was added]2-Formaldehyde (2.46g, 9.9mmol) was reacted at room temperature for 1 h. Sodium triacetoxyborohydride (5.72g, 27mmol) was added and reacted at room temperature for 6 hours. Filtration and organic phase with saturated NaHCO 3 And saturated NaCl solution, MgSO 4 Drying and spin-drying to obtain a crude product. Column chromatography gave a white solid, yield: 79 percent. The melting point is 92-94 ℃. 1 H NMR(400MHz,DMSO-d 6 )δ11.63(s,1H),7.99(d,J＝2.5Hz,1H),7.75(d,J＝9.0Hz,1H),7.50–7.46(m,1H),7.42(d,J＝2.3Hz,1H),7.35(d,J＝8.3Hz,2H),7.05(d,J＝8.3Hz,2H),6.78–6.70(m,1H),6.40–6.31(m,2H),3.65(s,3H),3.12(m,4H),2.73(m,2H),2.19(m,6H),1.97(d,J＝12.7Hz,2H),1.39(t,J＝6.3Hz,2H),0.94(s,6H).
Preparation of 2- ((1H-pyrrolo [2,3-b ] pyridinyl-5-yl) oxy ] -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) benzoic acid:
methyl 2- ((1H-pyrrolo [2, 3-b)]Pyridyl-5-yl) oxy]-4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl)]-2-yl) methyl) piperazin-1-yl) benzoate (4.09g, 7mmol) in THF/MeOH/H 2 To a mixed solvent of 1: 3: 1(60mL) was added KOH (1.57g, 28mmol), and the mixture was refluxed for 6 hours. The organic phase was evaporated and the pH adjusted to 2 with 6M HCl. And (3) filtering to obtain a product, wherein the yield is as follows: 89 percent. Melting point 249-. 1 H NMR(400MHz,DMSO-d 6 )δ11.57(s,1H),7.96(s,1H),7.60(d,J＝8.5Hz,1H),7.42(s,1H),7.35(m,3H),7.05(d,J＝7.8Hz,2H),6.64(d,J＝8.6Hz,1H),6.33(s,1H),6.29(s,1H),3.03(s,4H),2.73(s,2H),2.29–2.11(m,6H),1.96(s,2H),1.39(t,2H),0.93(s,6H).
Preparation of 2- (((1H-pyrrolo [2,3-b ] pyridinyl-5-yl) oxy ] -4- (4- (((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- ((5- (dimethylamino) naphthalen-1-yl) sulfonyl) benzamide (11):
2- ((1H-pyrrolo [2, 3-b) under ice bath]Pyridyl-5-yl) oxy]-4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl)]Isobutyl chloroformate (0.21mL, 1.65mmol) and N-methylmorpholine (0.20mL, 1.65mmol) were added to a solution of (0.86g, 1.5mmol) in DMF (30mL) and reacted for 30 minutes to give a mixed anhydride. To a solution of 5- (dimethylamino) naphthalene-1-sulfonamide (0.41g, 1.65mmol) in DMF (30mL) was added NaH (0.09g, 1.5mmol), and the mixture was stirred at 80 ℃ for 30 minutes. The solution is added into mixed anhydride and reacted for 8h at 80 ℃. Stopping heatingAfter cooling to room temperature, the reaction was poured into water (120mL) and extracted three times with ethyl acetate. And (3) combining organic phases, evaporating to remove the solvent, and purifying a crude product by using a column chromatography method to obtain a white solid, wherein the yield is as follows: 60%, melting point 175-. 1 H NMR(400MHz,CDCl 3 )δ10.40(s,1H),9.06(s,1H),8.61(d,J＝7.3Hz,1H),8.57(d,J＝8.6Hz,1H),8.21(s,1H),8.13(d,J＝8.6Hz,1H),7.85(d,J＝8.8Hz,1H),7.70(s,1H),7.61(t,J＝8.0Hz,1H),7.49(s,1H),7.22(d,J＝7.6Hz,2H),7.07(d,J＝7.5Hz,1H),6.90(d,J＝7.3Hz,2H),6.59(s,1H),6.48(d,J＝9.0Hz,1H),5.96(s,1H),3.02(s,4H),2.86(s,6H),2.72(s,2H),2.24–2.08(m,6H),1.95(s,2H),1.40(t,J＝6.0Hz,2H),0.92(s,6H). 13 C NMR(101MHz,CDCl 3 )δ160.68,158.29,154.53,151.08,145.33,144.37,141.05,135.68,134.21,132.77,131.41,130.89,130.42,128.74,128.65,128.51,128.02,127.30,127.19,126.30,122.34,119.99,119.76,117.17,113.85,108.19,108.02,100.46,99.67,59.27,51.17,45.89,44.37,34.28,28.68,28.16,27.13,24.54.HRMS(AP-ESI)m/z,Calcd for C 45 H 47 ClN 6 O 4 S,([M+H] + ):803.3141,found:803.3122.
Example 22 preparation of- (((1H-pyrrolo [2,3-b ] pyridinyl-5-yl) oxy ] -4- (4- (((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- (((10-ethyl-9-oxo-9, 10-dihydroacridin-2-yl) sulfonyl) benzamide (12):
Example 32- ((1H-pyrrolo [2,3-b ] pyridin-5-yl) oxy ] -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- (quinolin-8-yl) benzamide (13) preparation:
2- ((1H-pyrrolo [2, 3-b)]Pyridyl-5-yl) oxy]-4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl)]-2-yl) methyl) piperazin-1-yl) benzoic acid (0.57g, 1mmol) was dissolved in DMF (30mL), DIPEA (0.19mL, 1.1mmol) and HATU (0.46g, 1.2mmol) were added and reacted at room temperature for 30 minutes. 8-aminoquinoline (0.15g, 1.1mmol) was added and reacted at 60 ℃ for 6 h. After cooling to room temperature, the reaction mixture was poured into water (90mL) and extracted 3 times with ethyl acetate (60 mL). Removing the solvent, purifying the crude product by column chromatography to obtain a white solid, wherein the yield is as follows: 61 percent. Melting point 114-116 ℃. 1 H NMR(400MHz,CDCl 3 )δ12.24(s,1H),9.40(s,1H),8.99(d,J＝7.7Hz,1H),8.47(s,2H),8.28(d,J＝8.9Hz,1H),8.07(d,J＝8.1Hz,1H),7.80(s,1H),7.55(t,J＝7.9Hz,1H),7.44(d,J＝8.2Hz,1H),7.40(s,1H),7.30(dd,J＝8.1,4.2Hz,1H),7.24(s,2H),6.94(d,J＝7.6Hz,2H),6.73(d,J＝9.0Hz,1H),6.52(s,1H),6.33(s,1H),3.14(s,4H),2.80(s,3H),2.29(s,4H),2.20(s,2H),1.98(s,2H),1.43(t,J＝6.1Hz,2H),0.95(s,6H). 13 C NMR(101MHz,CDCl 3 )δ163.35,157.78,154.68,152.61,148.00,147.10,145.66,142.15,139.12,137.05,135.99,135.94,133.58,131.97,121.33,120.99,120.41,119.53,116.88,114.25,109.59,102.46,101.40,100.00,60.41,52.48,47.48,47.03,38.63,35.37,29.23,28.20,25.64.HRMS(AP-ESI)m/z,Calcd for C 42 H 41 ClN 6 O 2 ,([M+H] + ):697.3052,found:697.3033.
Example 42 preparation of- ((1H-pyrrolo [2,3-b ] pyridinyl-5-yl) oxy ] -4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl ] -2-yl) methyl) piperazin-1-yl) -N- (4-methyl-2-oxo-2H-benzofuran-7-yl) benzamide (14):
compound 14 was prepared as compound 13, white solid, yield: and 47 percent. Melting point 136-. 1 H NMR(400MHz,CDCl 3 )δ9.34(s,1H),8.71(d,J＝4.2Hz,1H),8.41(d,J＝8.4Hz,1H),8.21(d,J＝8.4Hz,2H),7.66(s,1H),7.40(dd,J＝10.9,8.2Hz,2H),7.24(,J＝7.4Hz,,2H),6.93(d,J＝7.3Hz,2H),6.61(d,J＝9.0Hz,1H),6.49(s,1H),6.16(s,1H),3.19(s,4H),2.78(s,2H),2.26(m,5H),2.19(m,2H),1.98(s,2H),1.62(s,2H),1.43(t,J＝5.8Hz,2H),0.95(s,1H). 13 C NMR(101MHz,CDCl 3 )δ162.70,160.13,156.57,151.54,146.73,145.80,142.10,141.18,136.75,135.36,135.12,134.59,131.96,129.71,129.35,129.08,128.28,126.53,120.56,120.14,107.89,102.61,101.63,101.29,60.32,52.24,47.01,46.74,35.36,29.23,28.19,25.62.HRMS(AP-ESI)m/z,Calcd for C 43 H 42 ClN 5 O 4 ,([M+H] + ):728.2925,found:728.2974.
Example 5 experiment for measuring fluorescence Properties of Compounds 11 to 14
The compound is prepared into concentrated stock solution with the concentration of 10mM by using dimethyl sulfoxide, and the concentrated stock solution is diluted by using PBS buffer solution to obtain solutions with different concentrations; the fluorescence spectra of the compounds at different concentrations and in different solvents were determined using a fluorescence spectrophotometer to explore the fluorescence properties of the probes, diluted to 1 μ M in different organic solvents. FIGS. 1-4 show the spectroscopic properties of compounds 11-14, respectively. The result shows that the compound of the invention has good fluorescence characteristic and is suitable for biomedical fluorescence detection.
EXAMPLE 6 fluorescence Properties determination after Co-incubation of Compounds 11 and 12 with Bcl-2 protein
FIG. 5 shows that the fluorescence intensity was increased when 1 μ M of Compound 11 was incubated with different concentrations of Bcl-2 protein, and the fluorescence was increased as the concentration of Bcl-2 protein was higher, while the fluorescence intensity returned to the initial state after the addition of Bcl-2 inhibitor ABT-199. The compound 11 and other proteins do not generate fluorescence enhancement phenomenon after incubation, and the above experiment result shows that the compound 11 can specifically recognize Bcl-2 protein, and the higher the concentration of the Bcl-2 protein is, the stronger the fluorescence signal released by the compound 11 is. The results show that the compound 11 is a high-energy and high-selectivity fluorescent tool, and can be used for early diagnosis of cancer, sorting of tumor cells and high-throughput screening of Bcl-2 protein inhibitors.
Furthermore, as shown in FIG. 6, the fluorescence intensity of Compound 12 was enhanced after incubation with Bcl-2 protein, whereas the fluorescence intensity of Compound 12 was not significantly changed after incubation with Mcl-1 protein or Bovine Serum Albumin (BSA). The above results indicate that compound 12 also specifically recognizes Bcl-2 protein and its fluorescence intensity increases with increasing concentration of incubated Bcl-2 protein. Example 7 application of Compound 11 in imaging of renal carcinoma cells ACHN (cells highly expressing Bcl-2 protein) and HUVECs (cells lowly expressing Bcl-2 protein) in normal tissue cells.
Cultured ACHN cells and HUVECs cells were seeded into confocal dishes at 37 deg.C and 5% CO 2 After culturing for 12h in the constant-temperature incubator, the culture medium is discarded, the cells are washed by PBS buffer solution for three times, then 1 mu M solution of the compound 11 is added, and after incubation for 20min in the dark at room temperature, imaging is carried out by a Zeiss Axio Observer A1 fluorescence microscope.
As shown in FIG. 7, ACHN was labeled with green fluorescence in renal cancer cells highly expressing Bcl-2 protein, whereas green fluorescence was not observed in HUVECs in normal tissue cells lowly expressing Bcl-2 protein. The above results indicate that compound 11 is capable of selectively labeling tumor cells.
EXAMPLE 8 Compound 11 for sorting tumor cells
We constructed a tissue model containing tumor cells and normal cells (tissue model 1: kidney cancer cells ACHN and normal tissue cells HUVECs are mixed; tissue model 2: human leukemia cells HL-60 and normal tissue cells HUVECs are mixed). The tissue model and 1 μ M compound 11 were incubated for 20min under dark conditions, and then the fluorescence properties of the cells were analyzed by flow cytometry. The results show (FIG. 8) that in both tissue models, tumor cells released stronger fluorescence after co-incubation with compound 11 due to high expression of Bcl-2 protein, while normal cells emitted weaker fluorescence due to lower content of Bcl-2 protein. Therefore, the tumor cells and the normal cells are successfully separated into two clusters according to the strength of the released fluorescence, so that the separation of the tumor cells and the normal cells is realized.
After the compound 11 of the present invention is incubated with a cell sample, the change of the fluorescence intensity of the sample is detected, and if the fluorescence enhancement phenomenon occurs, the cell is a tumor cell. Accordingly, the compound 11 of the present invention can be used for detecting and sorting tumor cells.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty, based on the technical solutions of the present invention.
Claims (9)
1. A Bcl-2 small-molecule fluorescent probe is characterized by having a structure shown in the following structural general formula I:
wherein R is 1 Represents a hydrogen atom, a halogen atom, C 1-6 Alkyl or C 1-6 A halogenated alkyl group,
R 2 、R 3 each independently represents a hydrogen atom, a halogen atom, C 1-6 Alkyl or C 1-6 A halogenated alkyl group,
R 4 represents a hydrogen atom, C 1-6 Alkyl or C 3-8 A cycloalkyl group, which is a cyclic alkyl group,
x is N or CH ═ CH,
r is a fluorescent group.
2. The Bcl-2 small molecule fluorescent probe of claim 1, wherein R is 1 Represents a hydrogen atom, a fluorine atom, a chlorine atom, C 1-4 Alkyl or C 1-4 A halogenated alkyl group,
R 2 、R 3 are independent of each otherRepresents a hydrogen atom, a halogen atom, C 1-4 Alkyl or C 1-4 A halogenated alkyl group,
x is CH (CH-CH),
R 4 represents a hydrogen atom, C 1-4 Alkyl or C 3-6 A cycloalkyl group,
r is 5-dimethylamino-1-sulfonyl naphthalene, 8-aminoquinoline, 8-hydroxyquinoline, 7-amino-4-methylcoumarin, 7-hydroxy-4-methylcoumarin, 8-sulfonylamino quinoline, 2-sulfonylamino-10-ethyl acridone, 4- (N-benzopyrrolinone) -benzene sulfonamide and tetraphenyl ethylene derivatives.
3. The Bcl-2 small molecule fluorescent probe of claim 1 or 2, selected from the group consisting of compounds having the following structural formula:
4. the method for preparing a Bcl-2 small molecule fluorescent probe according to claim 1 or 2, which comprises:
3, 3-dimethylcyclohexanone reacts with phosphorus oxychloride and N, N-dimethylformamide to generate an intermediate 2; the intermediate 2 reacts with p-chlorobenzoic acid to generate an intermediate 3; 4-chloro-2-fluorobenzoic acid is protected by methyl ester to obtain an intermediate 4, then the intermediate 4 reacts with Boc-piperazine to generate an intermediate 6, then a nucleophilic substitution reaction is carried out to obtain an intermediate 7, and the intermediate 8 is obtained after the Boc protecting group is removed; then the intermediate 8 is subjected to reductive amination reaction to obtain an intermediate 9, and the intermediate 10 is obtained after hydrolysis; reacting the intermediate 10 with fluorophores with different structures to obtain a compound with a general formula I;
the reaction route is as follows:
wherein R, R1, R2, R3 and R4 are as defined in formula I above;
reaction reagents and reaction conditions: (a) phosphorus oxychloride, N-dimethylformamide, dichloromethane; (b) [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride, substituted aryl boric acid or heterocyclic boric acid, 1, 4-dioxane and water; (c) refluxing acetyl chloride and methanol; (d) palladium acetate, cesium carbonate, toluene, 80 ℃; (e) sodium hydride, N-dimethylformamide, 140 ℃; (f) hydrogen chloride saturated ethyl acetate, dichloromethane; (g)3, 1, 2-dichloroethane, sodium triacetoxyborohydride; (h) LiOH, tetrahydrofuran/water; (i) R-NH 2 Isobutyl chloroformate, N-methylmorpholine, tetrahydrofuran; or R-NH 2 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane.
5. Use of a Bcl-2 small molecule fluorescent probe according to any one of claims 1-3 for the preparation of a reagent for the specific recognition and quantitative determination of Bcl-2 proteins.
6. The use of a Bcl-2 small molecule fluorescent probe of any one of claims 1-3 in the preparation of a reagent for achieving high throughput screening of a Bcl-2 protein inhibitor and in the preparation of a reagent for detecting inhibition of Bcl-2 protein.
7. Use of the Bcl-2 small molecule fluorescent probe according to any one of claims 1 to 3 for preparing a labeling and sorting reagent for tumor cells highly expressing Bcl-2 protein.
8. The use of the Bcl-2 small-molecule fluorescent probe as defined in any one of claims 1-3 for preparing a reagent for the early diagnosis of malignant tumors related to Bcl-2 protein overexpression.
9. The use of the Bcl-2 small-molecule fluorescent probe according to claim 8, wherein the malignant tumor related to Bcl-2 protein overexpression is pancreatic cancer, lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, multiple myeloma and leukemia.
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