CN115043833B - Bcl-2 fluorescent probe and preparation method and application thereof - Google Patents

Bcl-2 fluorescent probe and preparation method and application thereof Download PDF

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CN115043833B
CN115043833B CN202110254474.9A CN202110254474A CN115043833B CN 115043833 B CN115043833 B CN 115043833B CN 202110254474 A CN202110254474 A CN 202110254474A CN 115043833 B CN115043833 B CN 115043833B
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方浩
梁涛
杨新颖
李佳
侯旭奔
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Shandong University
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Abstract

The invention relates to a Bcl-2 small molecule fluorescent probe, a preparation method and application thereof, and the structure of the compound is shown as a general formula I, wherein R is as follows 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 of the invention can be used for preparing high-throughput screening reagents of Bcl-2 protein inhibitors, early diagnosis of cancers and marking and sorting reagents of tumor cells.

Description

Bcl-2 fluorescent probe and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicines, in particular to a Bcl-2 small molecular fluorescent probe and a preparation method thereof, and application thereof in Bcl-2 protein specific quantitative detection, bcl-2 inhibitor screening, cancer detection and cell sorting.
Background
Bcl-2 family proteins are an important class of signal molecules, and can be classified into pro-apoptotic proteins (Bax, bak, etc.), anti-apoptotic proteins (Bcl-2, mcl-1, bcl-X, etc.) according to structure and function L Etc.) and BH3-only proteins (Bad, bid, bim, noxa, etc.). The complex interaction between the Bcl-2 family pro-apoptotic proteins and anti-apoptotic proteins can accurately regulate and control the endogenous apoptosis pathway mediated by mitochondria, and has important significance for the development of multicellular organisms and the maintenance of the homeostasis of the in-vivo environment.
Under normal physiological conditions, the expression levels of anti-apoptotic proteins and pro-apoptotic proteins of the Bcl-2 family are maintained in dynamic balance to achieve accurate regulation of endogenous apoptotic pathways, but certain pathological stimuli or genetic mutations can cause imbalance in the expression of anti-apoptotic proteins and pro-apoptotic proteins, leading to a range of diseases such as autoimmune diseases, neurodegenerative diseases, cancer, and the like. In addition, overexpression 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. Studies show that over-expression of Bcl-2 family anti-apoptosis proteins (such as Bcl-2, mcl-1 and the like) is a main cause of tumor cell apoptosis escape, and over-expression of Bcl-2 family anti-apoptosis proteins has become one of tumor cell markers, and over-expression of Bcl-2 family anti-apoptosis proteins is found in various tumor cells such as breast cancer, lung cancer, prostate 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 level of anti-apoptotic proteins in the cells.
At present, several Bcl-2 protein fluorescent probes have been developed, but their low affinity to target proteins and poor selectivity have greatly limited their use. In addition, existing Bcl-2 fluorescent probes are also unable to detect Bcl-2 protein expression levels 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, and the series of probes show extremely high affinity and selectivity to Bcl-2 proteins and are successfully applied to detection of Bcl-2 protein expression in living cells.
Disclosure of Invention
The invention provides a Bcl-2 small molecule fluorescent probe, a preparation method thereof and application thereof in preparing a labeling reagent and a sorting reagent for Bcl-2 protein quantitative determination, early cancer diagnosis and tumor cells.
The specific technical scheme is as follows:
1. 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 atomSon, C 1-6 Alkyl or C 1-6 A haloalkyl group, a halogen atom,
R 2 、R 3 independently represent a hydrogen atom, a halogen atom, C 1-6 Alkyl or C 1-6 A haloalkyl group, a halogen atom,
R 4 represents a hydrogen atom, C 1-6 Alkyl or C 3-8 A cycloalkyl group,
x is N or ch=ch,
r is a fluorescent group.
Preferably, the method comprises the steps of,
the R is 1 Represents hydrogen, fluorine, chlorine, C 1-4 Alkyl or C 1-4 A haloalkyl group, a halogen atom,
R 2 、R 3 independently represent a hydrogen atom, a halogen atom, C 1-4 Alkyl or C 1-4 A haloalkyl group, a halogen atom,
x is ch=ch and is preferably selected from the group consisting of,
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-sulfonylquinoline, 2-sulfonylamino-10-ethyl acridone, 4- (N-benzopyrrolidone) -benzenesulfonamide, or a tetraphenyl derivative.
It is further preferred that the composition comprises,
the Bcl-2 small molecule fluorescent probe is selected from the group consisting of compounds having the following structural formula:
2. preparation method of Bcl-2 small molecule fluorescent probe
The preparation method of the Bcl-2 small molecule fluorescent probe comprises the following steps:
3, 3-dimethyl cyclohexanone reacts with phosphorus oxychloride and N, N-dimethylformamide to generate an intermediate 2; intermediate 2 reacts with p-chlorophenylboronic acid to generate 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 nucleophilic substitution reaction is carried out to obtain an intermediate 7, and the intermediate 8 is obtained after removing Boc protecting groups; then intermediate 8 is subjected to reductive amination reaction to obtain intermediate 9, and intermediate 10 is obtained after hydrolysis; reacting the intermediate 10 with fluorophores with different structures to obtain a compound shown in a general formula I;
the reaction route is as follows:
wherein R, R, R2, R3 and R4 are defined as the general formula I;
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) acetyl chloride, methanol, reflux; (d) palladium acetate, cesium carbonate, toluene, 80 ℃; (e) sodium hydride, N-dimethylformamide, 140 ℃; (f) ethyl acetate saturated with hydrogen chloride, 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-aza-benzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine, dichloromethane.
3. Application of Bcl-2 small molecule fluorescent probe
The application of the Bcl-2 small molecule fluorescent probe in preparing a reagent for specifically recognizing and quantitatively determining Bcl-2 protein;
the Bcl-2 small molecule fluorescent probe is applied to preparation of a reagent for realizing high-throughput screening of Bcl-2 protein inhibitors and detecting and inhibiting Bcl-2 protein;
the Bcl-2 small molecule fluorescent probe is applied to the preparation of a labeling and sorting reagent for tumor cells with high Bcl-2 protein expression;
the Bcl-2 small molecule fluorescent probe is applied to preparation of early diagnosis reagents for related malignant tumors of Bcl-2 protein overexpression.
The Bcl-2 protein overexpression related malignant tumor in the application of the Bcl-2 small molecule fluorescent probe is pancreatic cancer, lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, multiple myeloma and leukemia.
The "halogen" as used herein refers to fluorine atom, chlorine atom, bromine atom, iodine atom, etc. Fluorine atoms and chlorine atoms are preferred.
"halo" as used herein means that any one of the atoms in the group that can be substituted is replaced with a halogen, and can be perhalogenated, i.e., the halogen atom replaces all of the positions in the group that can be substituted.
The invention is described as "C 1-6 Alkyl "means a straight-chain or branched 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. The invention is described as "C 1-4 Alkyl "refers to the above examples containing 1-4 carbon atoms.
The 3-8 membered cycloalkyl includes 3-8 membered saturated monocyclic cycloalkyl and 3-8 membered partially saturated monocyclic cycloalkyl. 3-8 membered saturated monocyclic cycloalkyl means that the monocyclic ring is a fully saturated carbocyclic ring, examples of which include, but are not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane, methylcyclohexane, dimethylcyclohexane, and the like. By 3-8 membered partially saturated monocyclic cycloalkyl is meant 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 "," C 3-6 Cycloalkyl groups "are specific examples containing 3 to 8 and 3 to 6 carbon atoms, respectively, in the following examples.
The invention has the beneficial effects that:
1. the probe has novel structure, extremely high affinity and selectivity to Bcl-2 protein, and has the characteristics of trace fluorescence probe, high efficiency and high specificity.
2. The Bcl-2 fluorescent probe can selectively mark tumor cells, and can realize early diagnosis of cancers and related diseases and sorting of tumor cells. The invention discloses an application of a Bcl-2 small molecular fluorescent probe in preparing a pharmaceutical preparation for screening Bcl-2 protein related malignant tumor, wherein the related malignant tumor is malignant tumor; pancreatic cancer, lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, multiple myeloma, and leukemia are preferred.
Drawings
FIG. 1 shows (A) absorption spectra of compound 11 (B) fluorescence excitation spectra of probe in PBS buffer at different concentrations (C) fluorescence emission spectra of probe in PBS buffer at different concentrations (D) fluorescence excitation spectra of probe in different solvents (E) fluorescence emission spectra of probe in different solvents.
Fig. 2 shows (a) absorption spectra (B) of compound 12 and fluorescence excitation spectra (C) of the probe in the PBS buffer solution at different concentrations and fluorescence emission spectra (D) of the probe in the PBS buffer solution at different concentrations and fluorescence emission spectra (E) of the probe in different solvents.
Fig. 3 shows (a) absorption spectrum (B) fluorescence excitation spectrum (C) of the probe in the PBS buffer solution at different concentrations of the compound 13, fluorescence emission spectrum (D) of the probe in the PBS buffer solution at different concentrations of the compound, and fluorescence emission spectrum (E) of the probe in different solvents.
Fig. 4 shows (a) absorption spectra of compound 14 (B) fluorescence excitation spectra of probe in PBS buffer solution at different concentrations (C) fluorescence emission spectra of probe in PBS buffer solution at different concentrations (D) fluorescence excitation spectra of probe in different solvents (E) fluorescence emission spectra of probe in different solvents.
FIG. 5 is a fluorescent property analysis of Compound 11 after co-incubation with Bcl-2 protein. (A) Fluorescence emission spectra after incubation of 2. Mu.M of Compound 11 with different concentrations of Bcl-2 protein (0.0125,0.025,0.05,0.075,0.1 and 0.125 mg/mL) for 20min at room temperature; (B) Fluorescence emission spectra after incubation of 2. Mu.M Compound 11, 2. Mu.M Compound 11+0.125mg/mL Bcl-2 protein, 2. Mu.M Compound 11+0.125mg/mL Bcl-2 protein+5. Mu.M ABT-199 (Bcl-2 protein inhibitor) for 20min at room temperature; (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 solution at room temperature; (D) Fluorescence intensity bar graph after incubation of 2. Mu.M Compound 11,0.125mg/mL Bcl-2 protein, 2. Mu.M Compound 11+0.125mg/mL Mcl-1 protein for 20min at room temperature.
FIG. 6 is a fluorescent property analysis of Compound 12 after co-incubation with Bcl-2 protein. (A) Fluorescence emission spectra of 1. Mu.M Compound 12 incubated with different concentrations of Bcl-2 protein (0.025,0.05,0.1,0.2 and 0.4 mg/mL) in buffer 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, 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, 1. Mu.M of Compound 12, 1. Mu.M of Compound 12+0.4mg/mL Bcl-2 protein, 1. Mu.M of Compound 12+0.4mg/mL Mcl-1 protein, 1. Mu.M of Compound 12+0.4mg/mL BSA. (D) Fluorescence intensity bar graph after incubation for 20min at room temperature with 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 of imaging (A1: ACHN cells, bright field imaging; A2: ACHN cells, fluorescence imaging; B1: HUVEC cells, bright field imaging; B2: HUVEC cells, fluorescence imaging) after incubation of 1. Mu.M compound 11 with ACHN and HUVEC cells, respectively.
FIG. 8 (A) shows the results of flow assays after 20min incubation of ACHN and HUVEC mixed cells with 1. Mu.M Compound 11 at room temperature; (B) Results of flow assays after incubation of HL-60 and HUVEC mixed cells with 1. Mu.M Compound 11 for 20min at room temperature.
Detailed Description
The above-described aspects of the present invention will be described in further detail with reference to the following embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention.
Example 12 preparation of chloro-4, 4-dimethylcyclohex-1-en-1-carbaldehyde:
n, N-dimethylformamide (1.16 mL,15 mmol) was dissolved in dichloromethane (30 mL) and POCl was added under ice-bath 3 (1.05 mL,11.25 mmol) was reacted at room temperature for 2h. 3, 3-dimethylcyclohexanone (1.04 mL,7.5 mmol) was then added and reacted at room temperature for 4h. The reaction solution was poured into ice water (120 mL) and extracted 3 times with methylene chloride. The organic phases were combined, mgSO 4 Drying, spin drying to obtain crude product, purifying by column chromatography to obtain colorless oily liquid, and yield: 76%. 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 charged 4-chlorophenylboronic acid (0.75 g,4.81 mmol), pd (dppf) Cl 2 (0.11 g,0.148 mmol). 1, 4-Dioxahexacyclic solution of 2-chloro-4, 4-dimethylcyclohex-1-ene-1-carbaldehyde (0.64 g,3.7 mmol) and K were added 2 CO 3 (1.33 g,9.62 mmol) in water, and nitrogen gas was purged three times and then reacted at 85℃for 6 hours. 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. Removal of Pd (dppf) Cl by filtration through a pad of celite 2 The organic phase is concentrated to obtain crude product. Column chromatography gave a colorless oil, yield: 78%. 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 (5.4 mL,75 mmol) was slowly added to anhydrous methanol (140 mL) and reacted for 30 min under ice-bath. 4-bromo-2-fluorobenzoic acid (7.01 g,30 mmol) was added to the solution and refluxed for 6 hours. The solvent was distilled off, and the crude product was washed with n-hexane to give a white solid, yield: 98%, melting point 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 (0.47 g,2 mmol), 1-Boc-piperazine (0.52 g,2.8 mmol), pd (OAc) 2 (0.0224 g,0.1 mol) and BINAP (0.125 g,0.2 mol), cs 2 CO 3 (0.7197 g,2.2 mol) was added to a two-necked flask, dried toluene (20 mL) was added, and after nitrogen substitution, the mixture was reacted at 80℃for 8 hours. The heating was stopped, cooled to room temperature, and the celite pad was filtered. The filtrate was dried by spin-drying. Recrystallizing the crude product with ethyl acetate to obtain white solid with yield: 81%, melting point 148-149 ℃. 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:
4- (3-fluoro-4- (methoxycarbonyl) phenyl) piperazine-1-carboxylic acid tert-butyl ester was dissolved in anhydrous N, N-dimethylformamide (120 mL) and 1H-pyrrolo [2, 3-b) was added]Pyridin-5-ol (2.1 g,15 mmol) and NaH (0.66 g,16.5 mmol) were replaced three times with nitrogen. The reaction was heated at 120℃for 8h. After the heating was stopped and cooled to room temperature, the reaction solution was poured into water (300 mL). Extracted three times with ethyl acetate (150 ml) and dried over anhydrous magnesium sulfate. Evaporating the solvent, purifying the crude product by using a column chromatography method to obtain white solid, and obtaining the yield: 76, melting point 193-194 ℃. 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 (4.5 g,10 mmol) was dissolved in ethyl acetate (60 mL) and HCl/EA (40 mL) was added. After stirring overnight, filtration was performed. To the filter cake was added ethyl acetate (100 mL) and saturated NaHCO 3 The mixed solution (100 mL) was stirred at room temperature for 1h and extracted three times with ethyl acetate. The organic phases were combined, anhydrous MgSO 4 Drying, spin drying the solvent to give a white solid, yield: 96%, melting point 146-148 ℃. 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 ] 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) benzoate:
2- ((1H-pyrrole [2, 3-b)]Pyridin-5-yl) oxy]Methyl-4- (piperazin-1-yl) benzoate (3.17 g,9 mmol) dissolved in 1, 2-dichloroethane (80 mL) 4-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [1, 1-biphenyl was added]-2-Formaldehyde (2.46 g,9.9 mmol) was reacted at room temperature for 1h. Sodium triacetoxyborohydride (5.72 g,27 mmol) was added and reacted at room temperature for 6 hours. Filtered, the organic phase was saturated with NaHCO 3 And saturated NaCl solution, mgSO 4 Drying, spin drying to obtain crude product. Column chromatography gave a white solid, yield: 79%. Melting point 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 ] 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) benzoic acid:
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 (4.09 g,7 mmol) THF/MeOH/H 2 To a mixed solvent of O=1:3:1 (60 mL), KOH (1.57 g,28 mmol) was added and the mixture was refluxed for 6 hours. The organic phase was distilled off and the ph=2 was adjusted with 6M HCl. Filtering to obtain the product, yield: 89%. Melting point 249-250 ℃. 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 ] 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- ((5- (dimethylamino) naphthalen-1-yl) sulfonyl) benzamide (11):
2- ((1H-pyrrolo [2, 3-b) under ice bath]Pyridinyl-5-yl) oxy]-4- (4- ((4 '-chloro-5, 5-dimethyl-3, 4,5, 6-tetrahydro- [ [1,1' -biphenyl)]Isobutyl chloroformate (0.21 mL,1.65 mmol) and N-methylmorpholine (0.20 mL,1.65 mmol) were added to a solution of-2-yl-methyl-piperazin-1-yl) benzoic acid (0.86 g,1.5 mmol) in DMF (30 mL) and reacted for 30 minutes to give the mixed anhydride. To a solution of 5- (dimethylamino) naphthalene-1-sulfonamide (0.41 g,1.65 mmol) in DMF (30 mL) was added NaH (0.09 g,1.5 mmol) and the mixture was stirred at 80℃for 30 min. The above solution was added to the mixed anhydride and reacted at 80℃for 8 hours. Heating was stopped, cooled to room temperature, the reaction was poured into water (120 mL) and extracted three times with ethyl acetate. The organic phases are combined, the solvent is distilled off, and the crude product is purified by column chromatography to obtain white solid with the yield: 60%, melting point 175-176 ℃. 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 ] 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- (((10-ethyl-9-oxo-9, 10-dihydro-acridin-2-yl) sulfonyl) benzamide (12):
preparation of compound 12 as compound 11, yield: 62%. Melting point 222-224 ℃. 1 H NMR(400MHz,CDCl 3 )δ10.24(s,1H),9.20(s,1H),9.17(s,1H),8.56(d,J＝8.5Hz,2H),8.26(s,1H),7.93(d,J＝9.0Hz,1H),7.80(d,J＝7.9Hz,1H),7.76(s,1H),7.62(d,J＝8.8Hz,1H),7.57(d,J＝8.6Hz,1H),7.45(s,1H),7.37(t,J＝7.5Hz,1H),7.22(d,J＝7.4Hz,2H),6.90(d,J＝7.6Hz,2H),6.57(s,1H),6.50(d,J＝9.0Hz,1H),5.97(s,1H),4.49(d,J＝7.0Hz,2H),3.49(s,2H),3.04(s,4H),2.73(s,2H),2.28–2.11(m,6H),1.95(s,2H),1.40(t,J＝6.5Hz,3H),0.93(s,6H). 13 C NMR(101MHz,DMSO-d 6 )δ176.56,173.29,164.44,158.44,154.97,146.98,145.92,143.99,142.45,141.63,135.88,135.38,134.84,132.59,132.45,131.30,130.46,129.30,129.19,128.53,128.27,128.12,127.32,122.90,122.52,120.91,120.26,118.54,117.02,116.71,109.16,102.75,100.44,60.11,52.48,47.04,46.77,41.54,35.27,29.32,28.37,25.60,12.76.HRMS(AP-ESI)m/z,Calcd for C 48 H 47 ClN 6 O 5 S,([M+H] + ):855.3090,found:855.3065.
Example 3 2- ((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) was prepared:
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 (0.57 g,1 mmol) was dissolved in DMF (30 mL), DIPEA (0.19 mL,1.1 mmol) and HATU (0.46 g,1.2 mmol) were added and reacted at room temperature for 30 min. 8-aminoquinoline (0.15 g,1.1 mmol) was added and reacted at 60℃for 6h. Cooled to room temperature, the reaction mixture was poured into water (90 mL) and extracted 3 times with ethyl acetate (60 mL). Removing the solvent, purifying the crude product by column chromatography to obtain white solid, and obtaining the yield: 61%. 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 4 2 preparation of- ((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- (4-methyl-2-oxo-2H-benzofuran-7-yl) benzamide (14):
compound 14 was prepared as compound 13, white solid, yield: 47%. Melting point 136-138 ℃. 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 experiments for determination of fluorescence Properties of Compounds 11-14
The compound is prepared into a concentrated stock solution with the concentration of 10mM by dimethyl sulfoxide, and the stock solution is diluted by PBS buffer solution to obtain solutions with different concentrations; the fluorescence characteristics of the probe were investigated by diluting 1. Mu.M solutions with different organic solvents and measuring the fluorescence spectra of the compounds at different concentrations and different solvents using a fluorescence spectrophotometer. FIGS. 1-4 are the spectroscopic properties of compounds 11-14, respectively. The results show that the compound of the invention has good fluorescence characteristics and is suitable for biomedical fluorescence detection.
Example 6 determination of fluorescence Properties after Co-incubation of Compounds 11 and 12 with Bcl-2 protein
FIG. 5 shows that incubation of 1. Mu.M Compound 11 with different concentrations of Bcl-2 protein resulted in an increase in fluorescence intensity, with the higher Bcl-2 protein concentration providing more fluorescence, and with the addition of the Bcl-2 inhibitor ABT-199, the fluorescence intensity was returned to its original state. The compound 11 is incubated with other proteins without fluorescence enhancement, and the experimental results show that the compound 11 can specifically identify Bcl-2 protein, and the higher the concentration of Bcl-2 protein, the stronger the fluorescence signal released by the compound 11. The results show that the compound 11 is a high-energy and high-selectivity fluorescent tool and can be used for early diagnosis of cancers, separation 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 did not change significantly 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 use of compound 11 in imaging of renal cancer cells ACHN (cells that highly express Bcl-2 protein) and normal tissue cells HUVECs (cells that lowly express Bcl-2 protein).
Inoculating cultured ACHN cells and HUVECs cells into confocal dish, respectively, at 37deg.C, 5% CO 2 After 12h incubation in a thermostatted incubator, the medium was discarded, the cells were washed three times with PBS buffer, 1. Mu.M solution of Compound 11 was added, incubated at room temperature for 20min in the absence of light and imaged with a Zeiss Axio Observer A1 fluorescence microscope.
As shown in FIG. 7, in renal cancer cells ACHN highly expressing Bcl-2 protein were labeled with green fluorescence, whereas in normal tissue cells HUVECs low expressing Bcl-2 protein, no green fluorescence was observed. The above results indicate that compound 11 is able to selectively label tumor cells.
Example 8 Compound 11 for sorting of tumor cells
We constructed a tissue model containing tumor cells and normal cells (tissue model 1: mixing kidney cancer cells ACHN with normal tissue cells HUVECs; tissue model 2: mixing human leukemia cells HL-60 with normal tissue cells HUVECs). The tissue model was incubated with 1. Mu.M Compound 11 for 20min under light-shielding conditions, and then the fluorescence properties of the cells were analyzed using a flow cytometer. The results show (FIG. 8) that in both tissue models, tumor cells highly expressed Bcl-2 protein, which released stronger fluorescence after co-incubation with Compound 11, whereas the Bcl-2 protein content in normal cells was lower, and the fluorescence emitted was weaker. Therefore, according to the intensity of fluorescence release, the tumor cells and the normal cells are successfully separated into two clusters, thereby realizing the separation of the tumor cells and the normal cells.
After incubating the compound 11 of the present invention with a cell sample, the change in fluorescence intensity of the sample is detected, and if a fluorescence enhancement phenomenon occurs, it is indicated that the cell is a tumor cell. Accordingly, compound 11 of the present invention can be used for detection and sorting of tumor cells.
While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that the invention is not limited to the particular embodiments, but is capable of numerous modifications and variations within the spirit and scope of the invention.

Claims (6)

1. A Bcl-2 small molecule fluorescent probe selected from the group consisting of compounds having the following structural formula:
。
2. the use of the Bcl-2 small molecule fluorescent probe of claim 1 in the preparation of a reagent for specific recognition and quantification of Bcl-2 protein.
3. The use of the Bcl-2 small molecule fluorescent probe of claim 1 in the preparation of a high throughput screening reagent for the realization of Bcl-2 protein inhibitors, for the preparation of a reagent for the detection of inhibition of Bcl-2 proteins.
4. The use of the Bcl-2 small molecule fluorescent probe of claim 1 for the preparation of a labeling and sorting reagent for tumor cells with high Bcl-2 protein expression.
5. The use of the Bcl-2 small molecule fluorescent probe of claim 1 for the preparation of an early diagnostic reagent for Bcl-2 protein overexpression-related malignancies.
6. The use of the Bcl-2 small molecule fluorescent probe of claim 5, wherein the Bcl-2 protein over-expression related malignancy is pancreatic cancer, lung cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, multiple myeloma, and leukemia.
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