CN108251104B - A kind of substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe and its application - Google Patents
A kind of substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe and its application Download PDFInfo
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Abstract
The invention discloses a kind of substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe and its applications.The general structure of the fluorescence probe such as formula is as follows:In general formula I, R1For all kinds of fluorogens；R2For halogen；General formula II, R3For all kinds of fluorogens；R4For halogen.Such fluorescent small molecule probe can be used as the research of the probe and physiology relevant to Bcl-2, pathology and related disease of identification Bcl-2 family protein；It can be used for Bcl-2 and inhibit agent high flux screening and its application in anti-tumor aspect；It can be used for the label of Bcl-2 family protein and its highly expressed tumour cell or tissue.In addition, the preparation method reaction condition of such compound is mild, raw material is cheap and easily-available, and operation and post-processing are simple.
Description
Technical field
The present invention relates to substituted indole -2- carboxylic acid derivates and its as Bcl-2 small-molecule fluorescent probe, with it in Bcl-
Application in the screening of 2 inhibitor activities, Cytotoxic evaluation and cell imaging, belongs to technical field of pharmaceuticals.
Background technique
B cell lymphoma/leukaemia -2 (B-cell leukemia/lymphoma-2, Bcl-2) protein family is cell
Important regulatory factor in apoptosis intrinsic pathway, plays important adjustment effect in Apoptosis access.According to structure with
Function is different, and Bcl-2 protein family can be divided into anti-apoptotic family protein and promote apoptosis family protein.In normal cell, promote
Apoptosis family protein and anti-apoptotic family protein heterodimerisation make cell keep normal growth state, had both avoided cell too early
Apoptosis avoids cell from being converted into tumour cell because apoptosis not occurring again.But in some malignant tumours, Bcl-2 protein family
Anti-apoptotic proteins member overexpression, causes general chemotherapy means that can not make tumour cell that apoptosis occur, to influence tumour
Therapeutic effect, generate drug resistance.Therefore, have become for the inhibitor research of Bcl-2 protein family anti-apoptotic member close
The research hotspot of anti-tumor drug over year.
More than 25 Bcl-2 protein family members are being had been found that in the mammalian body at present, these members are according to structure
Two subtribes: anti-apoptotic proteins and pro apoptotic protein can be divided into function difference.Anti-apoptotic proteins member includes Bcl-2, Bcl-
Xl, Mcl-1, Al, Bcl-w, Bcl-RAMBO and Boo.Subtribe member is able to suppress Apoptosis, most of (in addition to Mcl-1
And A1) all containing there are four Bcl-2 homologous region (Bcl-2homology, BH) BH1-BH4.Pro apoptotic protein can be divided into two Asias again
Family: the multizone pro apoptotic protein subfamily (Bak, Bax etc.) containing 2-3 conservative region and a conservative BH3 is contained only
The BH3-only albumen (Bad, Bik, Bid, Bim, Hrk, Bmf, Puma, Noxa etc.) in region.
Bcl-2 protein family is key regulator in apoptotic mitochondrial approach, penetrating by adjusting mitochondrial membrane
Property regulating cell apoptosis.Under normal circumstances, Bax and Bak and Bcl-2 protein family anti-apoptotic member (such as Bcl-2, Mcl-1)
It is combined with each other with the presence of inactive monomeric form.Inactive Bax is distributed mainly in cytoplasm, and Bak then mainly inlays
On mitochondrial outer membrane.After cell receives apoptotic signal, some BH3-only albumen (such as Bad, Noxa) it is competitive with
The hydrophobic binding domain of Bcl-2 anti-apoptotic member combines, and releases repressed Bax and Bak albumen.Hereafter, other BH3-
Only albumen (such as Bim, Puma) directly can make its activation with Bax and Bak protein-interacting.Bax the and Bak albumen of activation
Oligomerization occurs on mitochondrial outer membrane, opens cell permeability hole, membrane permeability increases and discharges the cells such as cytochrome c
Antiapoptotic factors induce cell apoptosis.Therefore, the Bcl-2 protein family anti-apoptotic member of tumour cell is inhibited to can promote tumour thin
The apoptosis of born of the same parents.Currently, there are about five class Bcl-2 inhibitor, wherein most has been in clinical research such as Gossypol and has been in I/
II phase clinic etc., but pass through clinical research and find that, due to the diversity of apoptotic pathways, partial inhibitor can not still make to swell
Apoptosis occurs for oncocyte, to influence the therapeutic effect of tumour, generates drug resistance, so as to cause such drug clinical application by
To considerable restraint.
Small-molecule fluorescent probe has the characteristics that quick, sensitive, high-throughput and is easy to automate in protein labeling and imaging
There is important role in technology, small-molecule fluorescent probe has been widely used for the important biomolecules such as protein, nucleic acid point at present
In biology and the pharmacology detection of son, there is weight to the development in the fields such as disease mechanisms discussion, clinical diagnosis and drug screening
The meaning wanted.But can Small-molecule probe exclusively in conjunction with target protein it is less, the shortage of design and synthesis method is
Principal element, there is presently no for studying the small-molecule fluorescent probe to interact between Bcl-2 family protein.In consideration of it, being
Deeper into the mechanism of action and the more effective inhibitor of discovery understood between Bcl-2 albumen, it would be highly desirable to which researching and developing one kind can specificity
In conjunction with the Small-molecule probe of Bcl-2 albumen.
Summary of the invention
In view of the above shortcomings of the prior art, the present invention provides a kind of substituted indole -2- carboxylic acids Bcl-2 small molecule fluorescent
Probe and preparation method thereof, optical activity, bioactivity and the application in pharmaceutical field.
To achieve the above object, the technical solution of the present invention is as follows:
A kind of substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe has following general structural formula:
In general formula I, R1For all kinds of fluorogens；R2For halogen；
General formula II, R3For all kinds of fluorogens；R4For halogen.
Preferably, the R1For naphthalenesulfonamide and AIE class fluorogen；R2For chlorine；R3For naphthalenesulfonamide fluorogen；R4
For chlorine；
It is highly preferred that substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe of the invention is selected from following structural formula
Compound:
The preparation method of the substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe L1, reaction process are as follows:
Preferably, specific steps are as follows:
(1) 4- chloroaniline generates diazonium salt in hydrochloric acid and sodium nitrite under the conditions of, diazonium salt again with 2- cyclopentanone formic acid second
Ester generates intermediate 2,
(2) intermediate 2 passes through and reacts to obtain intermediate 3 at ester,
(3) intermediate 3 obtains product 4 through cyclization reaction again,
(4) product 4 obtains intermediate 5 through deprotection reaction.
(5) compound 6 obtains compound 7 under the conditions of ammonia,
(6) key intermediate 5 and 7 is amide condensed reacts through de- ethyl ester again, then can obtain final product L1.
It is further preferred that the specific steps of the step (1) are as follows: hydrochloric acid is added into round-bottomed flask, and water is added, it
Afterwards, under condition of ice bath, 3- chloroaniline is added into reaction flask, and the aqueous solution of nitrous acid is gradually added into after five minutes.2- ring
Pentanone Ethyl formate is dissolved in EtOH, is then gradually dropped with KOH solution, after half an hour, the reaction solution of 3- chloroaniline by
It is gradually added into latter reaction's liquid, mixed reaction solution reacts 1h in 40 DEG C of oil baths.Reaction solution is concentrated to obtain crude product 2,
It is not purified directly to carry out next step reaction.
The hydrochloric acid, water: 3- chloroaniline, nitrous acid, 2- cyclopentanone Ethyl formate, EtOH and potassium hydroxide use ratio
For (7m, 1M) L:9mL:(1.27g, 10mmol): (1.38g, 20mmol): (2.5mL, 15mmol): (5.0g, 90mmol).
It is further preferred that the specific steps of the step (2) are as follows: compound 2 be dissolved in ethanol solution the concentrated sulfuric acid by by
Gradually it is added.Mixed reaction solution back flow reaction 2h, rear reaction solution restore to room temperature, and alcohol solvent is evaporated off and is extracted with ethyl acetate
It takes, obtains pure products 3 through column chromatography (petroleum ether: ethyl acetate=5:1) after dry concentration.
The use ratio of the compound 2, ethyl alcohol and the concentrated sulfuric acid is (3.3g, 10mmol): 25mL:3mL.
The product 3 be light yellow solid, yield 40%, mp:81-83 DEG C.1H-NMR(400MHz,CDCl3),δ
9.86(s,1H),7.40-7.39(m,1H),7.28(s,1H),7.23-7.22(m,1H),6.94-6.91(m,1H),4.36-
4.27 (m, 4H), 2.65-2.61 (m, 2H), 2.50-2.47 (m, 2H), 1.80-1.74 (m, 2H), 1.43 (t, J=8.0Hz,
3H), 1.37 (t, J=8.0Hz, 3H).
It is further preferred that the specific steps of the step (3) are as follows: intermediate 3 is dissolved in toluene, and p-methyl benzenesulfonic acid is added
Enter reaction solution reflux 12h.Cooling reaction solution back spin is extracted with ethyl acetate except solvent, and the oil that drying obtains after being spin-dried for is through recrystallizing
(n-hexane) obtains pure compound 4.
The intermediate 3, toluene, toluenesulfonic acid use ratio be (0.68g, 2mmol): 10mL:(0.58g,
3mmol)。
The product 4 be light yellow solid, white solid, yield 83%, mp:93-95 DEG C.1H-NMR(400MHz,
CDCl3), δ 9.91 (d, J=12.0Hz, 1H), 7.68 (d, J=8.0Hz, 1H), 7.39 (s, 1H), 7.15-7.12 (m, 1H),
4.46-4.42 (m, 2H), 4.19-4.09 (m, 2H), 3.76-3.72 (m, 1H), 3.42 (t, J=8.0Hz, 1H), 2.74-2.67
(m, 2H), 1.47-1.44 (m, 3H), 1.30 (t, J=8.0Hz, 1H), 1.24 (t, J=8.0Hz, 2H).
It is further preferred that the specific steps of the step (4) are as follows: compound 4 is added into round-bottomed flask, glacial acetic acid
It is added into.Dense HCl is added into then reaction solution and is heated to 80 DEG C afterwards.After three hours, water is added into there is white solid after quilt
Filter to obtain product 5.
The compound 4, glacial acetic acid, dense HCl, water use ratio be (0.32g, 1mmol): 3.5mL:0.5mL:
10mL。
The product 5 be white solid, yield 89%, mp:210-213 DEG C.1H-NMR(400MHz,CDCl3),δ
12.09 (s, 1H), 11.71 (s, 1H), 7.74 (d, J=8.0Hz, 1H), 7.42 (t, J=4.0Hz, 1H), 7.11-7.07 (m,
1H), 4.36-4.32 (m, 2H), 4.27 (t, J=8.0Hz, 2H), 2.53 (t, J=8.0Hz, 2H), 1.37 (t, J=8.0Hz,
3H)。
It is further preferred that the specific steps of the step (5) are as follows: in the methylene chloride that compound 6 is dissolved in, be passed through ammonia
Gas after 15min, stops reaction, and solvent, which is revolved, to be removed, and white is gradually precipitated and filters to obtain product 7.
The use ratio of compound 6, the methylene chloride is (0.5g, 1.8mmol): 10mL.
The product 7 be white solid, yield 99%, mp:210-213 DEG C.1H-NMR(400MHz,CDCl3),δ
8.43 (d, J=8.0Hz, 1H), 8.30 (d, J=8.0Hz, 1H), 8.05 (d, J=8.0Hz, 1H), 8.13 (d, J=8.0Hz,
1H), 7.63-7.56 (m, 4H), 7.26 (d, J=8.0Hz, 1H), 2.83 (s, 6H).
It is further preferred that the specific steps of the step (6) are as follows: intermediate 5 is dissolved in dry methylene chloride, in ice
Under the conditions of bath, DIEA is added into, and after ten minutes, HATU is added into, reaction solution become cloudy continue stir half an hour after, compound 7
It is added into.Reaction solution is extracted after being stirred overnight with methylene chloride, and dry rotation is except yellow oil is obtained after solvent, oil is through simple column
Product 8 is chromatographed to obtain, directly carries out next step reaction without secondarily purified.Important intermediate 8 is dissolved in ethyl alcohol, 50% NaOH
Liquid is added into reaction solution, and overnight, solvent is evaporated off for reaction at room temperature, and water occurs after being added into 1M HCl tune pH to neutrality
White solid, white solid filtering are simultaneously recrystallized to give pure final product L1 by ethyl alcohol and water.
The compound intermediate 5, methylene chloride, DIEA, HATU, compound 7, NaOH, water use ratio be
(0.15g, 0.5mmol): 10mL:(0.19g, 1.5mmol): (0.23g, 1.2mmol): (0.14g, 0.55mmol): 2mL:
10mL。
The product L1 be yellow solid, yield 41%, mp:158-161 DEG C.1H-NMR(400MHz,DMSO-d6),
δ 11.51 (s, 1H), 8.44 (d, J=8.0Hz, 1H), 8.32-8.27 (m, 1H), 8.18 (d, J=8.0Hz, 1H), 7.62-
7.52 (m, 3H), 7.34 (s, 1H), 7.22-7.15 (m, 1H), 7.02 (d, J=8.0Hz, 1H), 6.94 (d, J=12.0Hz,
1H), 3.08 (t, J=8.0Hz, 2H), 2.83 (s, 6H), 2.41 (s, 2H)13C-NMR(100MHz,DMSO-d6):δ173.34,
163.52,151.72,137.84,136.52,129.95,129.79,129.68,129.41,129.28,128.02,126.19,
125.82,123.91,122.40,121.56,120.08,119.57,115.27,112.03,45.52,38.29,20.06HRMS
(ESI)m/z calcd forC35H38BrN8O5S([M-H]-)761.1869；found 761.1867.
The preparation method of the substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe L2, reaction process are as follows:
Preferably, specific steps are as follows:
(1) intermediate 5 restores after condensation reaction and obtains compound 10.
(2) compound 10 is reacted with dansyl chloride reacts to obtain final product L2 through de- ethyl ester again.
It is further preferred that the specific steps of the step (1) are as follows:
A. intermediate 5 is dissolved in THF liquid, SOCl2With DMF by subsequent addition.Reaction solution is stirred 2 small at room temperature
When.Then, ammonia is passed into reaction solution.It is stirred 1 hour after ammonia saturation, solvent, which is removed, is added a large amount of water, and white is solid
Pure product 9 is obtained by filtration in body appearance.
B. compound 9 is dissolved in THF, and the THF liquid of borine is added under conditions of nitrogen protection.Mixed reaction solution is in room temperature
Under be stirred 16h, after reaction, be concentrated after a large amount of ethyl alcohol quenching reactions are added, subsequent HCl is added into back flow reaction 1h, instead
Answer and neutralized by caustic lye of soda after liquid and extracted with ethyl acetate, obtained after concentration after crude product through column chromatography (petroleum ether:
Ethyl acetate=10:1) whitish product 10 is obtained, next step reaction is directly carried out without secondarily purified.
Compound intermediate 5, THF, SOCl in the step a2, DMF use ratio be (0.30g, 1mmol): 15mL:
0.11mL:2d.
The product 9 be white solid, yield 100%, mp:228-230 DEG C.1H-NMR(400MHz,CDCl3),δ
11.67 (s, 1H), 7.75 (d, J=8.0Hz, 1H), 7.41-7.39 (m, 1H), 7.23 (t, J=8.0Hz, 1H), 7.13-7.06
(m,1H),6.74-6.71(m,1H),4.37-4.32(m,2H),3.24-3.16(m,2H),2.39-2.33(m,2H),1.38
(t, J=8.0Hz, 3H).
The use ratio of the THF liquid of compound 9, THF, borine in the step b, HCl are as follows: (0.58g, 2mmol): 5mL:
(8mL, 8mmol): (3M, 4mL).
It is further preferred that the specific steps of the step (2) are as follows:
A. product 10 is dissolved in THF, Et3N is added into.After stirring 0.5h, dansyl chloride is added into and is stirred overnight.Then,
THF solvent be evaporated off crude product through column chromatography (petroleum ether: ethyl acetate=7:1) obtain pure product 11.
B. product 11 is dissolved in dehydrated alcohol, and 50%NaOH solution is added into, and rear mixed liquor is stirred overnight at room temperature.Second
It, alcohol solvent, which is revolved, to be removed, and 15mL water is added into.Reaction solution is neutralized by 1M HCl solution, and white solid occurs and filtered
Crude product is recrystallized to give pure final product L2 through ethyl alcohol and water after crude product.
Product 10, THF, Et in the step a3N, dansyl chloride (0.13g, 0.5mmol): 20mL:(0.15mL,
1.05mmol): (0.15g, 0.525mmol)
The product 11 be yellow solid, yield 84%, mp:118-120 DEG C.1H-NMR(400MHz,CDCl3),δ
11.61 (s, 1H), 8.45 (d, J=8.0Hz, 1H), 8.32 (d, J=8.0Hz, 1H), 8.05 (d, J=8.0Hz, 1H), 7.94
(s, 1H), 7.60 (t, J=8.0Hz, 2H), 7.40-7.37 (m, 2H), 7.26 (d, J=8.0Hz, 1H), 6.99 (d, J=
8.0Hz,1H),4.28-4.23(m,2H),2.81(s,10H),1.56-1.53(m,2H),1.26-1.23(m,3H).
Compound 11, NaOH, water use in the step b are than for 70mg:1.5ml:15mL.
The product L2 be yellow solid, yield 86%, mp:70-73 DEG C.1H-NMR(400MHz,DMSO-d6),δ
10.96 (s, 1H), 9.20 (s, 1H), 8.42 (t, J=8.0Hz, 2H), 8.05 (dd, J1=8.0Hz, J2=4.0Hz, 1H),
7.61-7.53 (m, 2H), 7.31-7.28 (m, 2H), 7.24 (d, J=8.0Hz, 1H), 6.87 (dd, J1=8.0Hz, J2=
4.0Hz,1H),2.97-2.94(m,2H),2.81(s,6H),2.70-2.49(m,2H),1.54-1.47(m,2H).13C-NMR
(100MHz,DMSO-d6):δ163.51,151.79,136.63,136.53,129.81,129.53,129.49,128.70,
128.27,126.29,125.36,124.02,122.35,122.17,120.12,119.56,115.54,112.08,45.49,
42.95,31.12,21.69.HRMS(ESI)m/z calcd.for C35H38BrN8O5S([M-H]-)761.1869；found
761.1867.
The preparation method of the substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe L3, reaction process are as follows:
Preferably, specific steps are as follows:
(1) key intermediate 5 and propargylamine it is amide condensed 12,
(2) diphenyl-methane is reacted with 4- methyl benzophenone obtains product 16 through dehydration, bromo, nucleophilic substitution again,
(3) final product L3 is obtained through deprotection reaction after key intermediate 16 and 12 is reacted through Click.
It is further preferred that the specific steps of the step (1) are as follows: propargylamine (30mg, 0.55mmol) is added in one
In round-bottomed flask, in the dichloromethane solution that product 5 and 4-dimethylaminopyridine are dissolved in, EDCI is added under condition of ice bath.
Be stirred overnight, reaction solution extracted with methylene chloride, dry rotation except after solvent through column chromatography (petroleum ether: ethyl acetate=
2:1) obtain product 12.
The compound propargylamine, product 5,4-dimethylaminopyridine, dichloromethane solution, EDCI use ratio be
(30mg, 0.55mmol): (0.15g, 0.5mmol): (74mg, 0.6mmol): 100mL:(0.29g, 1.5mmol).
The product 12 be pink solid, yield 42%, mp:180-183 DEG C.1H-NMR(400MHz,DMSO-
d6), δ 11.93 (s, 1H), 11.68 (s, 1H), 7.73 (d, J=8.0Hz, 1H), 7.42-7.40 (m, 1H), 7.10-7.06 (m,
1H),4.38-4.32(m,2H),3.88-3.86(m,1H),3.81-3.79(m,1H),3.53-3.48(m,1H),3.26(t,J
=8.0Hz, 1H), 3.10-3.07 (m, 1H), 2.43-2.36 (m, 2H), 1.38 (t, J=8.0Hz, 3H)
It is further preferred that the specific steps of the step (2) are as follows:
A. dry THF liquid is cooled to 0 DEG C in N after being added into a two neck bottles2Under protective condition, n-BuLi quilt
Slowly inject.Stirred meeting, diphenyl-methane are dropped into.Mixed reaction solution is stirred 1h, 4- methyl benzophenone under the conditions of 0 DEG C
It is added into reaction solution.Subsequent reactions liquid restores to room temperature and is stirred overnight.End of reaction is largely saturated NH4Cl solution quilt
It is extracted with dichloromethane after quenching reaction is added, petroleum ether is added after being merged concentration in organic layer, white solid occurs and filters
Product 13.
B. compound 13 and p-methyl benzenesulfonic acid are added in the round-bottomed flask of 50mL, toluene is added.Reaction solution is refluxed 4h.
After reaction solution is cooling, extracted with n-hexane.Solvent is collected and is concentrated.Later, petroleum ether, which is added into, there is white solid
Filtered to obtain intermediate 14.
C. be added in the round-bottomed flask of 100mL 14, N- bromo-succinimide and benzoyl peroxide, subsequent CCl4
It is added into reflux 12h.When reaction terminates, a large amount of water are added into and are extracted with methylene chloride.Merge organic layer decompression to steam
Pure products 15 are chromatographed to obtain except column is carried out with n-hexane after solvent.
D. intermediate 15 and sodium azide are added in the two-neck bottle of 100mL, DMSO, vacuum nitrogen gas, in nitrogen is added
It is reacted overnight under gas shielded.It second day, is extracted after a large amount of water are added with a large amount of methylene chloride, after merging methylene chloride concentration
Column chromatography (petroleum ether: ethyl acetate=150:1) obtains pure product 16.
The use ratio of THF, n-BuLi, diphenyl-methane, 4- methyl benzophenone, petroleum ether in the step a is
10mL:(5mL, 2.5M): (1.01g, 10mmol): (1.63g, 8.3mmol): 10mL.
The product 13 be white solid, yield 86%, mp:138-141 DEG C.1H-NMR(400MHz,DMSO-d6),
δ 7.47 (t, J=8.0Hz, 5H), 7.40 (t, J=8.0Hz, 3H), 7.13-6.92 (m, 11H), 5.87 (s, 1H),
The use ratio of compound 13, p-methyl benzenesulfonic acid, toluene, petroleum ether in the step b is (0.5g, 10mmol):
0.01g:10mL:5mL.
The product 14 be white solid, yield 78%, mp:175-178 DEG C.1H-NMR(400MHz,DMSO-d6),
δ 7.15-7.09 (m, 9H), 6.98-6.93 (m, 8H), 6.85 (d, J=8.0Hz, 2H), 2.20 (s, 3H)
Intermediate 14, N- bromo-succinimide, benzoyl peroxide, CCl in the step c4Using than for
(0.7g, 2.0mmol): (0.40g, 2.2mmol): 0.005g:12mL.
The product 15 be white solid, yield 64%, mp:120-123 DEG C.1H-NMR(400MHz,DMSO-d6),
δ7.13-7.09(m,11H),7.03-6.98(m,8H),4.42(s,2H).
The use ratio of intermediate 15, sodium azide, DMSO in the step d is (0.21g, 0.5mmol): (0.048g,
7.5mmol): 10mL.
The product 16 be white solid, yield 50%, mp:105-108 DEG C.1H-NMR(400MHz,DMSO-d6),
δ7.04-7.02(m,9H),6.97-6.94(m,10H),4.18(s,2H).
It is further preferred that the specific steps of the step (3) are as follows:
A. intermediate 12 and intermediate 16 are added in 50mL eggplant type bottle, are added solvent tertiary butanol/water (2:1).Then, anti-bad
Hematic acid sodium solution and copper-bath are added into reaction solution.Muddy reaction solution is heated to 50 DEG C of reactions under the conditions of being protected from light
1h is then cooled to room temperature.Methylene chloride is added except solvent in rotation.After organic layer is collected and is concentrated under reduced pressure after drying, pass through
It is simple that column chromatographs to obtain crude product 17.Next step reaction is directly carried out without second of purifying.
B. 50%NaOH solution is added after being dissolved in EtOH in key intermediate 17, and after stirring 12h, reaction dissolvent is used after being removed
1M HCl adjusts pH and white solid occurs to neutrality, filters out white solid with second alcohol and water and is recrystallized to give final product L3.
Intermediate 12, intermediate 16, butanol/water, sodium ascorbate solution, copper-bath in the step a make
With than for (0.07g, 0.2mmol): (0.086g, 0.22mmol): 10.0mL:(10mL, 0.1M): (0.1M, 1.6mL).
The use ratio of intermediate 17, EtOH, NaOH in the step b are as follows: 0.10g:5mL:2ml
The product L3 be gray solid, yield 73%, mp:208-210 DEG C.1H-NMR(400MHz,DMSO-d6),
δ 11.34 (d, J=8.0Hz, 1H), 9.16 (s 1H), 8.87 (s 1H), 7.78 (d, J=12.0Hz, 1H), 7.61 (d, J=
8.0Hz, 1H), 7.35-7.30 (m, 1H), 7.15-7.02 (m, 11H), 6.98-6.93 (m, 9H), 5.45 (d, J=8.0Hz,
2H),5.43(dd,J1=16.0Hz, J2=8.0Hz, 2H), 3.56 (t, J=8.0Hz, 2H), 2.45 (t, J=8.0Hz, 1H),
1.91(s,1H).13C-NMR(100MHz,DMSO-d6):δ177.74,172.55,172.20,172.10,171.84,146.09,
145.91,143.55,143.46,141.33,140.44,137.11,135.99,134.61,134.57,131.39,131.04,
128.37,128.31,128.26,127.82,127.13,127.02,126.73,126.08,123.41,123.32,122.07,
120.08,119.51,111.88,45.07,37.23,34.71,21.62,21.24.HRMS(ESI)m/z calcd.for
C42H34ClN5O3([M-H]-)690.2277；found 690.2272.
The preparation method of the substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe L4, reaction process are as follows:
Preferably, specific steps are as follows:
(1) dansyl chloride reacts to obtain compound 23 with 3- amino -1- propyl alcohol,
(2) compound 23 and 21 prolong through light react compound 24 is gone ethyl ester to react to obtain final product L4 again.
It is further preferred that the specific steps of the step (1) are as follows: the round bottom that dansyl chloride is added into a 100mL is burnt
In bottle, methylene chloride is added, 3- amino -1- propyl alcohol is dissolved in Na2CO3It is added in reaction flask after solution, mixed reaction solution is acute
Strong stirring 1h.Then evaporating solvent under reduced pressure.Ethyl acetate/petroleum ether (ethyl acetate: petroleum ether)=1 is added into reaction flask:
2, white solid is filtered to obtain pure products 23.
The compound dansyl chloride, methylene chloride, 3- amino -1- propyl alcohol, Na2CO3, ethyl acetate/petroleum ether use
Than for (0.27g, 1.00mmol): 10mL:(0.11g, 1.50mmol): (5.4mL, 2M:10mL.
The product 23 be white solid, yield 84%, mp:118-120 DEG C.1H-NMR(400MHz,DMSO-d6),
δ 8.62 (d, J=8.0Hz, 1H), 8.64 (d, J=8.0Hz, 1H), 8.62 (d, J=8.0Hz, 1H), 7.67-7.64 (m, 1H),
7.46 (s, 2H), 6.85 (d, J=8.0Hz, 1H), 4.50 (s, 1H), 4.06 (s, 2H), 3.37 (s, 8H), 1.75 (s, 2H)
It is further preferred that the specific steps of the step (2) are as follows:
A. intermediate 23, compound 21 and triphenylphosphine are added separately in a two neck bottles.It injects under nitrogen protection
Tetrahydrofuran solvent, subsequent diethyl azodiformate are slowly injected into, and reaction solution is heated to 70 DEG C of back flow reactions for 24 hours.Reaction
End solvent, which is depressurized, to be evaporated off.By simple that column chromatographs to obtain crude product 24.It is directly carried out in next step without secondarily purified.
B. key intermediate 24 is dissolved in ethyl alcohol, and NaOH solution is added, is stirred overnight at room temperature.Solvent is removed within second day
Fall, be added 1M HCl solution adjust pH to neutrality after do not go out solid, with ethyl acetate carry out extraction concentrate ethyl alcohol and water into
The final product L4 that row recrystallizes pure.
The use ratio of intermediate 23, compound 21, triphenylphosphine, dicarboxylate in the step a be (0.25g,
1.1mmol): (0.31g, 1mmol): (0.35g, 1.32mmol): (0.25g, 1.43mmol)
NaOH solution is 1mL 50%NaOH in the step b.
The product L4 be yellow solid, yield 88%, mp:200-203 DEG C.1H-NMR(400MHz,DMSO-d6),
δ 13.07 (s, 1H), 8.45 (d, J=8.0Hz, 1H), 8.31 (d, J=8.0Hz, 1H), 8.05-7.99 (m, 2H), 7.71 (d, J
=8.0Hz, 1H), 7.62-7.57 (m, 2H), 7.33 (d, J=8.0Hz, 1H), 7.27 (d, J=8.0Hz, 1H), 7.21-7.18
(m, 1H), 7.16 (s, 1H), 4.43 (t, J=8.0Hz, 2H), 2.87-2.74 (m, 6H), 1.72-1.65 (m, 2H), 1.24 (s,
1H),1.18(s,1H).13C-NMR(100MHz,DMSO-d6)δ162.84,151.84,137.30,136.23,129.93,
129.53,129.49,129.47,128.85,128.37,126.67,125.31,125.06,124.04,121.69,119.43,
115.59,112.83,109.92,45.49,42.29,30.82.HRMS(ESI)m/z calcd for C24H24ClN3O4S([M-
H]-)484.1098；found 484.1103.
Substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe of the invention can be used for Bcl-2 albumen and its high table
The label of the tumour cell or tissue that reach.
Substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe of the invention can directly compound of reaction to Bcl-2's
Inhibitory activity, the high flux screening that can be used for Bcl-2 inhibitor and its application in antitumor evaluation.
Substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe of the invention can be used as identification Bcl-2s albumen
The application of probe and Bcl-2 albumen in physiology, pathology and related disease.
Beneficial effects of the present invention:
1. it is novel to respond group, selectivity is good.
2. the preparation method reaction condition of probe of the present invention is mild, raw material is cheap and easily-available, and operation and post-processing are simple.
3. probe molecule bioactivity of the invention is strong, high with the affinity of Bcl-2 family protein, high sensitivity.
4. the label that probe of the invention can be used for Bcl-2 albumen and its highly expressed tumour cell or tissue.
5. probe of the invention can directly compound of reaction to the inhibitory activity of Bcl-2, can be used for the height of Bcl-2 inhibitor
Flux screening and its application in antitumor evaluation.
6. probe and Bcl-2 albumen that probe of the invention can be used as identification Bcl-2 albumen are in physiology, pathology and correlation
Have broad application prospects in disease.
Detailed description of the invention
Fig. 1 probe molecule L1 (A, 10 μM) or L2 (B, 10 μM) in HeLa cell has no inhibitor gossypol (10 μM), and there are items
Imaging (A, B:HeLa cell under part；C:HEK293 cell；1,3,5: light field；2,4,6: probe molecule green channel) camera lens
Amplification factor: 63 ×.
Fig. 2 probe molecule L4 (5 μM) HeLa cell in the case where there is no inhibitor gossypol (5 μM) existence condition imaging (A,
B:HeLa cell；C:HEK293 cell；1: light field；2: probe molecule green channel) amplification factor of camera lens: 63 ×.
Fig. 3 probe L1 (10 μM), L3 (10 μM)-L4 (5 μM) are with HeLa cell in the gossypol whether there is or not same concentrations in room temperature
Under be protected from light be incubated for 30min after through Flow cytometry measure streaming result (curve 1: blank control；Curve 2: probe adds same concentration to press down
Preparation；Curve 3: probe).
Fig. 4: HE dyeing (A): mouse tumor tissue slice；(B): mouse normal tissue sections；
Fig. 5: immunohistochemical experiment (A): tumor tissues immunohistochemistry (white light)；(C): mouse tumor tissues immunohistochemistry GFP
Channel shooting；(B): mouse normal tissue immunohistochemistry (white light)；(D): the shooting of the mouse normal tissue channel immunohistochemistry GFP.
Fig. 6: the fluorescence probe imaging experiment under the channel GFP: A and C is probe L1 (10 μM) and L3 (10 μM) mouse tumour
Imaging of tissue；The C and D points of mouse tumor tissues for probe L1 (10 μM) and gossypol (10 μM) and L3 (10 μM) and gossypol (10 μM) at
Picture；E and F is respectively probe L1 (10 μM) and L3 (10 μM) mouse imaging normal tissue.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
The optically active measurement of embodiment 1
Table 1: the optical signature of probe molecule
Note: all of above optical property measures in the phosphate buffer of PH=7.4.
Embodiment 2: the measurement of bioactivity
It with gossypol (Gossypol) for positive drug, is tested using FP or TR-FRET, measures fluorescent probe molecule and Bcl-
The combination activity experiment of 2 family proteins, the results are shown in Table 2.The activity and positive control of probe molecule L1, L3, L3 of synthesis
Medicine gossypol activity is suitable, and the activity of probe L4 is low compared with positive control drug gossypol activity.
Table 2: the affinity of probe molecule and Bcl-2 family protein
Embodiment 3: application of the probe molecule in Bcl-2 high expression and low expression HEK293 cell imaging
Using probe molecule L1 and L3-L4 as research object, investigate its application in cell imaging, select Hela cell for
Positive cell selects HEK293 for negative cells, under the same conditions, inhibitor gossypol is added as negative control.Specific step
Suddenly are as follows: Hela cell and DMEM culture medium of the HEK293 cell containing 10% fetal calf serum；In 5%CO2Air and 37 DEG C
It is cultivated in environment, before imaging, by cell inoculation in being copolymerized in burnt capsule, cultivates 12-24h, culture medium is sopped up, with being free of
The culture medium of serum washed once, and be separately added into probe L1-L4 (culture medium without serum is prepared, and concentration is 1 μM)；Identical item
Under part, another capsule is separately added into probe L1-L4, and (serum-containing media is not prepared, and L1 and L3 concentration are 10 μ with inhibitor gossypol
M, gossypol concentration are 10 μM；L4 concentration is 5 μM, and gossypol concentration is 5 μM) make negative control, it is incubated for, culture medium is then sucked out, washes
It washs once, is imaged with Zeiss Axio Observer A1.
Imaging results are as shown in Figures 1 and 2, and under negative cells comparison, probe molecule can mark Bcl-2 highly expressed
Hela cell, probe L1 and L3-L4 have broad application prospects in the research of Bcl-2 physiology, pathology and related disease.
Embodiment 3: probe and intracellular protein interaction inquiry experiment
We select to be lost experiment to probe into the interaction in probe molecule and cell between albumen, we use cell strain
For HeLa cell, before experiment, by kind after the cell centrifugation for being in exponential increase, in being lost in pipe, (every pipe > 500,000 are thin
Born of the same parents), experimental setup blank control is that probe is not added, and experimental group only adds probe and negative control group (inhibitor with concentration to be added
Gossypol and same concentration probe), streaming experiment is carried out using flow cytometer.
Streaming experimental result (see Fig. 3) display, compares blank control group, and HeLa cell is colored after probe is added, when adding
After entering inhibitor, cell is colored degree decline and illustrates that our probes are that Competition occurs with inhibitor, it may be said that bright probe
It is consistent with inhibitor action target spot is Bcl-2 family protein.
Embodiment 4: imaging applications of the probe in tumour and normal slice.
We select active preferable probe L1 and L3 to carry out mouse tumor slicing experiment, in order to observe the tumour position of slice
It sets, we have carried out HE dyeing and immunohistochemical experiment, the following Fig. 4 of experimental result and Fig. 5 institute to tumor biopsy and normal slice
Show, according to shown knub position is sliced, we have carried out fluorescence imaging experiments using probe L1 and L3.Experimental result such as Fig. 6 institute
Show, probe L1 and L3 can with the high-incidence position of marked tumor and with normal tissue or the phase in the case where there is the competition of inhibitor gossypol
Than, it is only stronger with the tumor biopsy fluorescence intensity of probe, these results indicate that perhaps our probe can be used for clinical tumor
Diagnosis.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to invention protection scope
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
It is still within the scope of the present invention to make the creative labor the various modifications or changes that can be made.
Claims (4)
1. a kind of substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe, which is characterized in that the Small-molecule probe is tool
Just like the compound of flowering structure formula:
。
2. substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe as described in claim 1 be used to prepare Bcl-2 and its
Application in the marker of highly expressed tumour cell or tissue.
3. substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe as described in claim 1 is in the height of Bcl-2 inhibitor
Application in flux screening.
4. spy of the substituted indole -2- carboxylic acids Bcl-2 small-molecule fluorescent probe as described in claim 1 as identification Bcl-2
The application of needle.
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