CN108690033A - The fluorescence probe and its preparation method and application of the molecule of pharmaceutical activity containing flavonoids - Google Patents
The fluorescence probe and its preparation method and application of the molecule of pharmaceutical activity containing flavonoids Download PDFInfo
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- CN108690033A CN108690033A CN201710217368.7A CN201710217368A CN108690033A CN 108690033 A CN108690033 A CN 108690033A CN 201710217368 A CN201710217368 A CN 201710217368A CN 108690033 A CN108690033 A CN 108690033A
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- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The present invention relates to fluorescence probe fields, disclose a kind of fluorescence probe and its preparation method and application of the molecule of pharmaceutical activity containing flavonoids, the fluorescence probe of the present invention is indicated by general formula X-L-Y, wherein X is the flavonoids pharmaceutical activity structure provided by following formula (1);Y is the fluorophor provided by following formula (2);Coupled structures L isOr, n is the integer of 1-6;In formula (1), R1,R4,R5And R6It is each independently selected from the alkoxy that hydrogen, hydroxyl or carbon atom number are 1-6;R2Selected from hydrogen or hydroxyl;R3For oxygen.The fluorescence probe of the molecule of pharmaceutical activity containing flavonoids of the present invention has specific target tropism, the also proliferation inhibition activity with higher tumour or cancer cell.
Description
Technical field
The present invention relates to a kind of fluorescence probes and its preparation method and application of the molecule of pharmaceutical activity containing flavonoids.
Background technology
Radix scutellariae (Scutellaria baicalensis) is a kind of traditional flavonoids Chinese medicine for inflammation, detumescence, is had
Many pharmacological actions such as anti-inflammatory, detumescence, antiviral and anticancer.Radix scutellariae is as natural products, complicated component, currently, science
Family still can not determine that the approach of the molecular mechanism of action and biological function that generate drug effect, resulting problem are always to restrict
The bottleneck of natural products class clinical drug application.
Fluorescence microscopy is the analytical technology newly risen in recent years, in environmental science, medicine, pharmacy and biology etc.
Field is widely used, especially in medicine and field of biology.Fluorescence probe believes the chemistry such as intermolecular interaction
Breath is converted into fluorescence signal and feeds back to the external world whole process " visualization ", certain substance such as drug etc. is made to be distributed and make in the cell
It is usually monitored in real time using fluorescence probe with mode.Therefore, the fluorescence probe of design specific target tropism is most important.
Invention content
The purpose of the present invention is to provide a kind of spies of the fluorescence of molecule of pharmaceutical activity containing flavonoids with specific target tropism
Needle and its preparation method and application, the fluorescence probe for containing flavonoids pharmaceutical activity molecule also have higher tumour or cancer thin
The proliferation inhibition activity of born of the same parents.
The present inventor has found by further investigation, provided by the present invention to contain flavones by what general formula X-L-Y was indicated
The fluorescence probe of class pharmaceutical activity molecule has special mitochondria and lysosome-targeting, this contains flavonoids pharmaceutical activity point
The fluorescence probe of son can act on to targeting mitochondria and lysosome in tumour or cancer cell, also, also have compared with
The proliferation inhibition activity of high tumour or cancer cell, the proliferation inhibition activity of the tumour or cancer cell that show is substantially not
Become, while as excellent fluorescence probe, it may have preferable medical value and market prospects.
One aspect of the present invention provides a kind of fluorescence probe of the molecule of pharmaceutical activity containing flavonoids as a result, wherein the fluorescence is visited
Needle is indicated by general formula X-L-Y, wherein X is the flavonoids pharmaceutical activity structure provided by following formula (1);Y is by following formula (2)
The fluorophor of offer;Coupled structures L is N is the integer of 1-6;
In formula (1), R1,R4,R5And R6It is each independently selected from the alkoxy that hydrogen, hydroxyl or carbon atom number are 1-6;R2It is selected from
Hydrogen or hydroxyl;R3For oxygen.
Second aspect of the present invention provides the preparation method of the fluorescence probe of the above-mentioned molecule of pharmaceutical activity containing flavonoids, wherein
This approach includes the following steps,
1) compound of structure shown in formula (2) withIt carries out amide condensed anti-
It answers, obtains the midbody compound that general formula L-Y is indicated, wherein n is the integer of 1-6;
2) make the midbody compound that general formula L-Y is indicated carry out Mannich with the compound of structure shown in formula (1) to react,
Obtain the fluorescence probe for the molecule of pharmaceutical activity containing flavonoids that general formula X-L-Y is indicated.
Third aspect present invention provides the fluorescence probe of the above-mentioned molecule of pharmaceutical activity containing flavonoids or above-mentioned preparation method
Application of the fluorescence probe of the obtained molecule of pharmaceutical activity containing flavonoids in preparing the drug for the treatment of tumour or cancer.
Through the above technical solutions, the fluorescence probe of the molecule of pharmaceutical activity containing flavonoids provided by the invention has well
Mitochondria and lysosome-targeting, the fluorescence probe for containing flavonoids pharmaceutical activity molecule can act on to targeting tumour or
Mitochondria in cancer cell and lysosome;Also, containing the flavonoids drug provided by the present invention indicated by general formula X-L-Y is lived
Property molecule the fluorescence probe also proliferation inhibition activity with preferable tumour or cancer cell.
Description of the drawings
Attached drawing is to be used to provide further understanding of the present invention, an and part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is fluorescence probe shown in 2 Chinese style of test case (3-1-2) structure in A549 (left side) and HeLa (right side) cell
Laser co-focusing fluorescence imaging figure.
Fig. 2 is fluorescence probe and cell membrane dyestuff DIO and nucleus dyestuff shown in 2 Chinese style of test case (3-1-2) structureLive 647RedLaser co-focusings of the Reagent in A549 (left side) and HeLa (right side) cell is glimmering
Light image.
Fig. 3 is that fluorescence probe shown in 2 Chinese style of test case (3-1-2) structure exists with mitochondrial dye Rhodamine123
Laser co-focusing fluorescence imaging figure in A549 (left side) and HeLa (right side) cell.
Fig. 4 is fluorescence probe and lysosome dyestuff DND-26 shown in 2 Chinese style of test case (3-1-2) structure on A549 (left side)
With the laser co-focusing fluorescence imaging figure in HeLa (right side) cell.
Fig. 5 is that fluorescence probe shown in 2 Chinese style of test case (3-1-2) structure exists with mitochondrial dye Rhodamine123
The overlapping degree interpretation of result figure of same two-dimentional dimension in A549 (left side) and HeLa (right side) cell.
Fig. 6 is fluorescence probe and lysosome dyestuff DND-26 shown in 2 Chinese style of test case (3-1-2) structure on A549 (left side)
With the overlapping degree interpretation of result figure of same two dimension dimension in HeLa (right side) cell.
Fig. 7 is that fluorescence probe and flavonoids pharmaceutical activity molecule shown in 2 Chinese style of test case (3-1-2) structure are thin to HeLa
The proliferation inhibition activity result figure of born of the same parents.
Specific implementation mode
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
Term " C used in this specification1-6Alkoxy " can be straight-chain or branched.Such as methoxyl group, second can be enumerated
Oxygroup, propoxyl group, different oxygen propyl group, butoxy, isobutoxy, sec-butoxy, tert-butoxy, amoxy, hexyloxy etc..
In the present invention, such asArbitrary site that can be on ring etc. the substituent R in similar structure is taken
Generation, multiple sites that can also be on ring are replaced, and when multiple sites are replaced, the substituent R in each site can
With identical or different.
In addition, in the present invention, " fluorescence probe of the molecule of pharmaceutical activity containing flavonoids is indicated by the following general formula X-L-Y " is
Referring to, the fluorescence probe of the molecule of pharmaceutical activity containing flavonoids can be one kind in the compound indicated by the following general formula X-L-Y,
Can be two or more in the compound indicated by the following general formula X-L-Y.
The fluorescence probe of the molecule of pharmaceutical activity containing flavonoids provided by the invention by general formula X-L-Y indicate, wherein X be by
The flavonoids pharmaceutical activity structure that following formula (1) provides;Y is the fluorophor provided by following formula (2);Coupled structures L isN is the integer of 1-6;
In formula (1), R1,R4,R5And R6It is each independently selected from the alkoxy that hydrogen, hydroxyl or carbon atom number are 1-6;R2It is selected from
Hydrogen or hydroxyl, R3For oxygen.
Preferably, R1,R4,R5And R6It is each independently selected from the alkoxy that hydrogen, hydroxyl or carbon atom number are 1-3;R2It is selected from
Hydrogen or hydroxyl;R3For oxygen;N is 1,2,3 or 4.
It is highly preferred that R1,R4,R5And R6It is each independently selected from the alkoxy that hydrogen, hydroxyl or carbon atom number are 1-3;R2Choosing
From hydrogen or hydroxyl;R3For oxygen;N is 2,3 or 4.
It is further preferred that R1,R4,R5And R6It is each independently selected from hydrogen, hydroxyl, methoxy or ethoxy;R2Selected from hydrogen
Or hydroxyl;R3For oxygen;N is 2,3 or 4.
In the preferred embodiment of the present invention, the general formula X-L-Y is the chemical combination of structure shown in following formula (3)
Object,
In the preferred embodiment of the present invention, the general formula X-L-Y is the change of structure shown in following formula (3-1)
Object is closed,
In the preferred embodiment of the present invention, the general formula X-L-Y is structure shown in following formula (3-1-1)
The compound of structure shown in compound and formula (3-1-2),
In addition, in a particularly preferred embodiment of the present, the fluorescence of the molecule of pharmaceutical activity containing flavonoids
Probe is the compound of structure shown in the compound of structure and/or formula (3-1-2) shown in above-mentioned formula (3-1-1).
The fluorescence probe of the molecule of pharmaceutical activity containing flavonoids is the compound and formula of structure shown in above-mentioned formula (3-1-1)
Shown in (3-1-2) when the compound of structure (when being also the two mixture), for the chemical combination of structure shown in above-mentioned formula (3-1-1)
Mixed proportion both in the mixture of the compound of structure shown in object and formula (3-1-2) does not require particularly.Preferably, formula
The molar ratio of the compound of structure shown in the compound of structure shown in (3-1-1) and formula (3-1-2) is 1:0.2-5, more preferably
1:0.3-4;More preferably 1:0.4-3;More preferably 1:0.5-2;More preferably 1:0.8-1.5;More preferably 1:0.9-1.2;
Particularly preferably 1:1.
The compound of structure shown in above compound provided by the invention, especially formula (3-1) has water-soluble well
And stability, and preferable tumour or cancer cell proliferation inhibition activity.
The present invention also provides the preparation methods of the fluorescence probe of the above-mentioned molecule of pharmaceutical activity containing flavonoids, wherein the party
Method includes the following steps,
1) compound of structure shown in formula (2) withIt carries out amide condensed anti-
It answers, obtains the midbody compound that general formula L-Y is indicated, wherein n is the integer of 1-6;
2) make the midbody compound that general formula L-Y is indicated carry out Mannich with the compound of structure shown in formula (1) to react,
Obtain the fluorescence probe for the molecule of pharmaceutical activity containing flavonoids that general formula X-L-Y is indicated.
According to the present invention, commercially available product may be used in the compound of structure shown in formula (2), can also be conventional by this field
It synthesizes and obtains after method optimization, when synthesis obtains, such as bibliography (1) Anzalone, A.V. may be used;Wang,T.Y.;
Chen,Z.;Cornish,V.W.Angew.Chem.Int.Ed.Engl.2013,52,650;(2)Uddin,M.J.;Crews,
B.C.;Ghebreselasie,K.;Marnett,L.J.Bioconjug.Chem.2013,24,712;(3)Uddin,M.J.;
Method described in Marnett, L.J.Org.Lett.2008,10,4799..
The preparation side of the compound of structure shown in Ming Dynasty style (2) for by taking the preparation of the compound of structure shown in formula (2) as an example
Method, the preparation method of the compound of structure shown in formula (2) are preferably carried out using following reaction routes:
Wherein, LiAlH4Indicate that lithium aluminium hydride reduction, NaBH4 indicate that sodium borohydride, 1M indicate that 1mol/L, reflux are indicated back
Stream.
According to the present invention, the compound of structure shown in formula (2) is preferably the compound and formula (2- of structure shown in formula (2-1)
2) compound of structure shown in,
According to the present invention, in step 1) compound of structure shown in formula (2) with
Amide condensed reaction is carried out, the midbody compound that general formula L-Y is indicated is obtained, wherein n is the integer of 1-6;Preferably, n 1,
2,3 or 4;It is highly preferred that n is 2,3 or 4.
The present invention a preferred embodiment in, in step 1) compound of structure shown in formula (2) withAmide condensed reaction is carried out, obtains the midbody compound that general formula L-Y is indicated, wherein n 2;It is specific
Preparation method is preferably carried out using following reaction routes:
Wherein, EDCI indicates that 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride, HOBT indicate 1- hydroxy benzenes
And triazole, TEA indicate triethylamine.
According to the present invention, in step 1), the compound of structure shown in the formula (2) with
Dosage can be changed in wide range.Preferably, the compound of structure shown in formula (2) with
Mole dosage ratio be 1:2-2.5.
It is described it is amide condensed reaction may be used amide reaction in conventional use of solvent, such as can be dichloromethane,
It is one or more in tetrahydrofuran and dimethylformamide (DMF);Preferably dimethylformamide.These solvents preferably use
Anhydrous solvent.In addition, in the present invention, to the concentration of the compound of structure shown in the formula (2) in a solvent, there is no special
It limits, as long as the L-Y midbody compounds can be obtained, such as can be 0.01-0.2mmol/mL, preferably
0.01-0.02mmol/mL。
In the present invention, it is preferred to which the condition of the amide condensed reaction includes:Reaction temperature is 15-80 DEG C, when reaction
Between be 10-72h;It is highly preferred that the condition of the amide condensed reaction includes:Reaction temperature is 40-50 DEG C, and the reaction time is
24-48h。
According to the present invention, in step 2), make the midbody compound that general formula L-Y is indicated and structure shown in above-mentioned formula (1)
Compound carries out Mannich reactions, obtains the fluorescence probe for the molecule of pharmaceutical activity containing flavonoids that general formula X-L-Y is indicated.
Preferably, in formula (1), R1,R4,R5And R6It is each independently selected from the alcoxyl that hydrogen, hydroxyl or carbon atom number are 1-3
Base;R2Selected from hydrogen or hydroxyl;R3For oxygen;It is highly preferred that R1,R4,R5And R6Being each independently selected from hydrogen, hydroxyl or carbon atom number is
The alkoxy of 1-3;R2Selected from hydrogen or hydroxyl;R3For oxygen;It is further preferred that R1,R4,R5And R6It is each independently selected from hydrogen, hydroxyl
Base, methoxy or ethoxy;R2Selected from hydrogen or hydroxyl;R3For oxygen.
In one preferred embodiment of the invention, the compound of structure shown in the formula (1) is following formula (1-1) institute
Show the compound of structure,
In a particularly preferred embodiment of the present, the knot of pharmaceutical activity containing flavonoids of above-mentioned Formula X-L-Y expressions
Structure fluorescence probe is one or more in the compound of structure shown in following formula (3).
Preferably, the compound of structure shown in formula (3) is the compound of structure shown in following formula (3-1),
It is highly preferred that general formula X-the L-Y is shown in the compound of structure and formula (3-1-2) shown in following formula (3-1-1)
The compound of structure,
Above-mentioned formula (3-1) compound can be synthesized according to following routes.
According to the present invention, in step 2), compound of the present invention to structure shown in the midbody compound and formula (1)
Mole dosage ratio there is no particular limitation, divide as long as the pharmaceutical activity containing flavonoids that above-mentioned general formula X-L-Y is indicated can be obtained
The fluorescence probe of son, such as the midbody compound and the mole dosage ratio of the compound of structure shown in formula (1) they are 1:
0.8-1.3。
In addition, the solvent that the Mannich reactions may be used is, for example, methanol, ethyl alcohol, tetrahydrofuran etc..In described
Concentration of the intermediate compounds therefor in the solvent is not particularly limited, as long as containing for the general formula X-L-Y expressions can be obtained
Flavonoids pharmaceutical activity structure fluorescence probe, such as the compound of structure shown in formula (1) can be 0.001-
0.002mmol/mL, more preferably 0.0015-0.002mmol/mL.
Preferably, the condition of the Mannich reactions includes:Reaction temperature is 20-110 DEG C, reaction time 1-20h;
It is highly preferred that the condition of the Mannich reactions includes:Reaction temperature is 65-80 DEG C, reaction time 4-10h.
In order to obtain more pure product, the preparation method of the fluorescence probe of the molecule of pharmaceutical activity containing flavonoids of the invention
Can also include that recrystallization or chromatographic separation and purification are carried out to gained fluorescence probe using ethyl alcohol.
The present invention also provides the fluorescence probe of the above-mentioned molecule of pharmaceutical activity containing flavonoids or above-mentioned preparation method are made
Application of the fluorescence probe of the molecule of pharmaceutical activity containing flavonoids obtained in preparing the drug for the treatment of tumour or cancer.
The present invention is not particularly limited the type of above-mentioned tumour or cancer.Preferably, the tumour or cancer are
Lung cancer, cervical carcinoma or breast cancer.
The fluorescence probe of pharmaceutical activity containing the flavonoids molecule provided by the present invention indicated by general formula X-L-Y, has very
Good Mitochondrially targeted property and lysosome-targeting.Also, the fluorescence of the molecule of pharmaceutical activity containing flavonoids provided by the invention is visited
Needle in addition to have good mitochondria and it is lysosome-targeting other than, the also Proliferation Ability with higher tumour or cancer cell
Activity acts on various tumours with enabling to the fluorescence probe targeting of the molecule of pharmaceutical activity containing flavonoids or cancer is thin
Born of the same parents, so as to further study its relevant molecule mechanism of action and approach in vivo.
Also, the fluorescence probe of the molecule of pharmaceutical activity containing flavonoids provided by the invention, the tumour or cancer shown are thin
The proliferation inhibition activity of born of the same parents is basically unchanged, and as the fluorescence probe of targeting specific, while also having preferable drug action.
The present invention will be described in detail by way of examples below.
Preparation example 1
Preparation of this preparation example for the compound of structure shown in formula (2).
(1) 7- hydroxyl -2- quinolones (compound 1) 3.23g (20mmol) are added in 90mL tetrahydrofurans, ice bath stirs
It mixes down and is slowly added to 1.22g (32mmol) lithium aluminium hydride reduction, be heated to reflux, reaction is overnight.Reaction stops postcooling, is added immediately
Reaction is quenched 35.0ml saturated ammonium chloride solutions, is extracted with ethyl acetate, organic phase by saturated nacl aqueous solution into
After row washing, it is concentrated to give yellow solid 2.83g, yield 95%.ESI-MS(m/z):[M+H]+calcd.for C9H11NO,
150.09;found,150.25.
(2) by 7- hydroxyls -1,2,3,4- tetrahydroquinolines (compound 2) 2.50g (16.8mmol) is added to 65mL acetic acid, room
It is slowly added to 2.53g (67.2mmol) sodium borohydride under temperature stirring.When room temperature reaction raw material about consumes half, second is added portionwise
Aldehyde is until raw material all consumes.Most of acetic acid is removed, NaHCO is used3PH is adjusted to neutrality, filtering, recrystallization obtain light yellow
Solid 2.68g, yield 90%.ESI-MS(m/z):[M+H]+calcd.for C11H16NO,178.12;found,178.10.
By obtained 1- ethyls -7- hydroxyls -1,2,3,4- tetrahydroquinolines (compound 3) 1.50g of step (2)
(8.47mmol) is added to 60mL n-butyric acies, and 0.82g (4.24mmol) trimellitic acid is added as catalyst in the 3 drop concentrated sulfuric acids
Acid anhydride (compound 4), is heated to reflux 12h.It is cooled to room temperature, ether is added, dark red oil is precipitated, collect dark red oil
Object, and (CHCl is detached by silica gel column chromatography3:CH3OH=5:1-1:1) dark red solid (yield, is obtained:28%) it, i.e., obtains respectively
To Formula (2-1) the i.e. 5-isomer and formula (2-2) i.e. 6-isomer of structure shown in formula (2).
The structural formula of formula (2-1) and formula (2-2) is as follows:
5-isomer:1HNMR (400MHz, DMSO-d6)δ(ppm):8.16 (d, J=7.6Hz, 1H), 8.01 (d, J=
8Hz,1H),7.59(s,1H),6.44(s,2H),6.22(s,2H),3.42-3.38(m,4H),3.28-3.25(m,4H),
2.47-2.42 (m, 4H), 1.76 (m, J=6.4Hz, 4H), 1.12 (t, J=8Hz, 6H) .MALDI-TOF-MS (m/z):[M]+
calcd.for C31H31N2O5 +,511.22;found,511.28.
6-isomer:1HNMR (400MHz, DMSO-d6)δ(ppm):8.41(s,1H),8.24(dd,J1=8Hz, J2=
1.2Hz, 1H), 7.32 (d, J=7.6Hz, 1H), 6.47 (s, 2H), 6.26 (s, 2H), 3.43-3.38 (m, 4H), 3.30-3.27
(m, 4H), 2.47-2.43 (m, 4H), 1.77 (m, J=6.4Hz, 4H), 1.12 (t, J=8Hz, 6H)13CNMR (400MHz,
DMSO-d6)δ(ppm):MALDI-TOF-MS(m/z):[M]+calcd.for C31H31N2O5 +,511.22;found,511.29.
Preparation example 2
Preparation of this preparation example for the compound of structure shown in formula (6).
(1) by the compound of structure shown in the compound of structure shown in 400mg (0.78mmol) formula (2-1) and formula (2-2)
Mixture (mixing molar ratio be 1:1), 179.5mg (0.94mmol) 1- ethyls-(3- dimethylaminopropyls) carbodiimide
Hydrochloride (EDCI), 126.5mg (0.94mmol) 1- hydroxy benzo triazoles (HOBT) and 325.5 μ L triethylamines are added to
In the dichloromethane of 100mL dryings, 1~2h is stirred to react at 50 DEG C.Add 285 μ L 1,8- diamino -3,6- dioxas
Octane, sustained response is for 24 hours.Add water to terminate reaction, extracts, is concentrated to give the compound of structure shown in formula (6-1) and formula (6-2) institute
Show the mixture of the compound of structure, is dark red solid (0.20g, yield 40.3%).The taking-up portion from said mixture
It is detached, obtains the compound of structure shown in the compound of structure and formula (6-2) shown in formula (6-1), the data of formula (6-2)
As follows.
Formula (6-2):1HNMR (400MHz, DMSO-d6)δ(ppm):8.68 (s, 1H), 8.27 (d, J=8Hz, 1H), 7.52
(d, J=8Hz, 1H), 6.93 (d, J=8Hz, 2H), 6.68 (d, J=8Hz, 2H), 3.61-3.51 (m, 12H), 3.00 (t, J1
=J2=8Hz, 2H), 2.89 (s, 1H), 2.74-2.72 (m, 4H), 2.63-2.58 (m, 4H), 2.28-2.26 (m, J=4Hz,
2H),1.91-1.78(m,6H),1.22(t,J1=J2=8Hz, 6H)13CNMR (400MHz, DMSO-d6)δ(ppm):169.8,
158.5,151.9,151.5,147.5,146.1,136.4,130.1,128.0,127.5,127.0,119.8,119.7,
119.6,118.9,118.3,104.3,104.1,100.0,96.6,96.2,72.6,70.1,67.6,57.2,48.0,45.6,
38.1,37.6,35.6,34.5,28.4,27.3,25.6,23.0,22.0,16.2,10.9.MALDI-TOF-MS(m/z):[M]+
calcd.for C37H45N4O6 +,641.33;found,641.30.
Formula (6-2):MALDI-TOF-MS(m/z):[M]+calcd.for C37H45N4O6 +,641.33;found,641.30.
Embodiment 1
The present embodiment is used to illustrate the fluorescence probe and preparation method thereof of the molecule of pharmaceutical activity containing flavonoids of the present invention.
(1) 100mg (0.16mmol) preparation example 2 is obtained into knot shown in the compound of structure and formula (6-2) shown in formula (6-1)
Mixture and 9.60mg (0.32mmol) paraformaldehyde and 2.18mg (0.016mmol) ZnCl of the compound of structure2It is added to
100mL ethyl alcohol after reacting 30min, is added the compound of structure shown in 45.3mg (0.16mmol) formula (1-1), is reacted at 65 DEG C
10h.It is cooled to room temperature, removes ethyl alcohol, residue silica gel column chromatography detaches (CHCl3:CH3OH=10:1), obtained solid is with efficiently
Liquid chromatogram (HPLC) isolates and purifies, and respectively obtains structure shown in the compound of structure and formula (3-1-2) shown in formula (3-1-1)
Compound (19.8mg, yield 13.2% is obtained in the two) obtains ().
Formula (3-1-2):1HNMR (400MHz, CD3OD)δ(ppm):8.26 (d, J=8Hz, 1H), 8.07 (d, J=8Hz,
1H), 7.93 (d, J=8Hz, 2H), 7.87 (d, J=8Hz, 1H), 7.65 (s, 1H), 7.50-7.48 (m, 5H), 6.72 (d, J=
12Hz, 1H), 6.60 (s, 1H), 5.24 (t, J=4Hz, 2H), 3.89 (m, 4H), 3.57-3.48 (m, 13H), 3.39 (t, J=
6Hz, 1H), 2.91-2.87 (m, 1H), 2.78-2.76 (m, 1H), 2.60 (s, 1H), 2.56 (s, 1H), 2.09 (t, J=8Hz,
2H),1.93(q,J1=8Hz, J2=4Hz, 4H), 1.84-1.80 (m, 4H), 1.50 (t, J=8Hz, 2H), 0.80 (t, J=
8Hz,6H).13CNMR (600MHz, CD3OD)δ(ppm):182.8,178.0,166.9,164.1,162.4,161.8,161.6,
156.9,156.5,155.7,154.4,153.6,153.2,148.4,137.7,134.6,132.1,131.9,131.2,
131.1,129.4,129.0,128.3,127.9,127.1,126.7,126.0,125.3,123.7,113.5,112.8,
109.0,107.1,104.7,103.7,94.3,69.9,69.0,61.0,41.8,39.7,39.0,37.4,35.1,31.7,
27.1,26.7,25.5,22.3,20.7,19.9,16.2,13.1,10.1.MALDI-TOF-MS(m/z):[M]+calcd.for
C54H57N4O11 +,937.4;found,937.4.
Formula (3-1-1):MALDI-TOF-MS(m/z):[M]+calcd.for C54H57N4O11 +,937.4;found,
937.4.
Test case 1:To kinds of tumor cells proliferation inhibition test
Mtt assay detection probe cell proliferation inhibitory activity test, respectively by cell line A549 (human lung adenocarcinoma cell line),
HeLa (human cervical carcinoma cell strain) and MCF-7 (Breast cancer lines strain) are incubated at containing 10 volume %fetal bovine
In the DMEM culture mediums (Gibco, USA) of serum (FBS, Hyclone, USA).It is collected after 2-3 days thin in exponential phase
Born of the same parents are inoculated in 96 orifice plate (A549:The μ L of 2000 cells/wells/100;HeLa:The μ L of 4000 cells/wells/100;MCF-7:4000 cells/
The μ L of hole/100) culture for 24 hours, be then respectively adding gradient concentration compound (200,100,50,25,10,5,2.5, μM).It is identical
Volume containing 1%DMSO and be not added with the culture medium of probe and flavonoids pharmaceutical activity molecule and be set as experimental comparison group, only 1%
For the culture solution of DMSO without the blank control group that is set as of cell, each concentration does 5 parallel laboratory tests.Continue after cultivating 48h, inhales
The culture medium containing probe is abandoned, is washed one time with the PBS in 200 holes μ L/, the culture medium of 100 μ L MTT containing 0.5mg/mL is added per hole, after
Continuous culture 4h.The culture medium containing MTT is abandoned in suction, not siphon away adherent crystal, and the DMSO of 100 μ L is added per hole, is placed in shaking table
Upper 10-15min makes dissolution of crystals.Then tissue culture plate is moved into microplate reader, surveys absorbance value in 492nm, mtt assay detection is thin
The survival rate of born of the same parents and the bioactivity for comparing both probe and flavonoids pharmaceutical activity structure.Cell survival rate calculation formula is as follows
It is shown:Survival rate (%)=[(experimental group OD values-blank group OD values)/(control group OD values-blank group OD value)s ]× 100%.And
The IC that curve matching calculates compound is done using Origin 8.0 software inhibiting rates and concentration50Value.
Table 1
In table 1, the flavonoids pharmaceutical activity structure of structure shown in formula (1-1) is to three kinds of A549, HeLa and MCF-7 cell
The compound of structure shown in a little higher than formula of inhibitory activity (3-1-2) of cell Proliferation is three kinds thin to A549, HeLa and MCF-7 cell
The inhibitory activity of born of the same parents' proliferation, but as shown in fig. 7, after modification shown in the compound of structure and formula (1-1) shown in gained formula (3-1-2)
The inhibitory activity trend of three kinds of cell proliferations of flavonoids pharmaceutical activity structure pair of structure is consistent, this illustrates flavonoids drug
Bioactive molecule remains the flavones of structure shown in formula (1-1) after the compound of structure shown in formula (3-1-2) obtained by modification
The bioactivity of class pharmaceutical activity molecule.
Test case 2:Laser co-focusing fluorescence imaging is tested
(1) cell imaging is analyzed
A-549 the and HeLa cell suspending liquids of 1mL are inoculated in the burnt ware of copolymerization, cell density is about 1 × 104A/mL,
12h is cultivated at 37 DEG C keeps its adherent.The compound of structure shown in the formula (3-1-2) of 2 μM (0.2%) is distinguished in 37 DEG C and cell
It is incubated 2h, 6h and 12h, without the compound of structure shown in formula (3-1-2) and only cell is as a control group.Remove drug containing
Culture medium, PBS solution wash three times, colourless basal medium DMEM is added.The burnt ware of copolymerization is moved into laser scanning copolymerization
It is imaged on the sample stage of focusing microscope.The excitation wavelength of laser scanning co-focusing micro-imaging is chosen to be 559nm, launch wavelength
570-670nm.It is opened with FV10-ASW 3.1Viewer softwares and handles obtained image, as shown in Figure 1.
(2) cell common location imaging analysis
A-549 the and HeLa cell suspending liquids of 1mL are inoculated in the burnt ware of copolymerization, cell density is about 1 × 104A/mL,
12h is cultivated at 37 DEG C keeps its adherent.The compound of structure shown in the formula (3-1-2) of 2 μM (0.2%) is distinguished in 37 DEG C and cell
It is incubated 2h, 6h and 12h, without the compound of structure shown in formula (3-1-2) and only cell is as a control group.Remove drug containing
Culture medium, PBS solution wash three times.Then selected cell membrane dyestuff DIO (5 μ L/mL) and nucleus dyestuff is addedLive 647RedReagent (2 drop), 15-20min is incubated at 37 DEG C, is washed again with PBS solution
It washs and colourless basal medium DMEM is added three times.The burnt ware of copolymerization is moved on the sample stage of laser scanning co-focusing microscope
Imaging.The excitation wavelength of laser scanning co-focusing micro-imaging is chosen to be 559nm, 488nm and 635nm, launch wavelength point respectively
It Wei not 570-615nm, 500-545nm and 650-750nm.It is opened and is handled obtained with FV10-ASW 3.1Viewer softwares
Image, as shown in Figure 2.Afterwards according to core, film positioning result, A-549 the and HeLa cells of inoculation 1mL suspend in the burnt ware of copolymerization
Liquid, cell density are about 1 × 104A/mL, culture 12h keeps its adherent at 37 DEG C.Formula (3-1-2) institute of 2 μM (0.2%)
Show that the compound of structure is incubated 2h, 6h and 12h respectively at 37 DEG C with cell, without the compound of structure shown in formula (3-1-2) and
Only cell is as a control group.The culture medium of drug containing is removed, PBS solution is washed three times.Then selected line grain is added
Body dyestuff Rhodamine123 (500nM) and lysosome dyestuffGreen DND-26 (500nM), 37
DEG C be incubated 15-20min, washed with PBS solution colourless basal medium DMEM is added three times again.The burnt ware of copolymerization is moved to
It is imaged on the sample stage of laser scanning co-focusing microscope.The excitation wavelength of laser scanning co-focusing micro-imaging is chosen to be respectively
559nm and 488nm, launch wavelength are respectively 570-615nm and 500-545nm.It is opened with FV10-ASW 3.1Viewer softwares
And obtained image is handled, as shown in Figure 3-4;And fluorescence probe and mitochondrial dye and lysosome dyestuff are in same two dimension
The overlapping degree of dimension, as seen in figs. 5-6.
The result of laser co-focusing fluorescence imaging is as shown in Figs 1-4, wherein Fig. 1 is respectively shown in formula (3-1-2) structure
Laser co-focusing fluorescence imaging figure of the fluorescence probe in A549 cells (left side) and HeLa cells (right side), Fig. 2 is respectively formula (3-
1-2) fluorescence probe, DIO shown in structure andLive 647RedReagent is in A549 cells
The laser co-focusing fluorescence imaging figure of common location in (left side) and HeLa cells (right side), Fig. 3 are respectively shown in formula (3-1-2) structure
Fluorescence probe and the laser of mitochondrial dye Rhodamine123 common locations in A549 cells (left side) and HeLa cells (right side) are total
Confocal fluorescence image, Fig. 4 are respectively that fluorescence probe and lysosome dyestuff DND-26 shown in formula (3-1-2) structure are thin in A549
The laser co-focusing fluorescence imaging figure of common location in born of the same parents (left side) and HeLa cells (right side).Fig. 5-6 is respectively fluorescence probe and line grain
The overlapping journey of body dyestuff and lysosome the dyestuff same two-dimentional dimension of common location in A549 cells (left side) and HeLa cells (right side)
Degree, by the comparison of Fig. 1-4, high-definition can find out, point of the fluorescence probe shown in formula (3-1-2) structure in cell
It is furnished with certain regionality, it is hardly be overlapped with cell membrane and nucleus dyestuff, illustrate that it is distributed in cytoplasm;And pass through
Mitochondrial dye and lysosome dyestuff common location result are it is found that fluorescence probe and mitochondrial dye signal be completely overlapped and lyase
Body dye signal partly overlaps;It can also be apparent from from Fig. 5-6, fluorescence probe and mitochondrial dye signal be completely overlapped,
It partly overlaps with lysosome dye signal, the two all illustrates one of action target spot or target spot of the molecule of pharmaceutical activity containing flavonoids
For mitochondria and lysosome.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In the skill of the present invention
In art conception range, technical scheme of the present invention can be carried out a variety of simple variants, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, belongs to
Protection scope of the present invention.
Claims (10)
1. a kind of fluorescence probe of the molecule of pharmaceutical activity containing flavonoids, which is characterized in that the fluorescence probe is by general formula X-L-Y tables
Show, wherein X is the flavonoids pharmaceutical activity structure provided by following formula (1);Y is the fluorophor provided by following formula (2);
Coupled structures L isN is the integer of 1-6;
In formula (1), R1,R4,R5And R6It is each independently selected from the alkoxy that hydrogen, hydroxyl or carbon atom number are 1-6;R2Selected from hydrogen or
Hydroxyl;R3For oxygen.
2. fluorescence probe according to claim 1, wherein R1,R4,R5And R6It is former to be each independently selected from hydrogen, hydroxyl or carbon
Subnumber is the alkoxy of 1-3;R2Selected from hydrogen or hydroxyl;R3For oxygen;N is 2,3 or 4.
3. fluorescence probe according to claim 2, wherein R1,R4,R5And R6It is each independently selected from hydrogen, hydroxyl, methoxyl group
Or ethyoxyl;R2Selected from hydrogen or hydroxyl;R3For oxygen;N is 2,3 or 4.
4. according to the fluorescence probe described in any one of claim 1-3, wherein the general formula X-L-Y is following formula (3) institute
Show the compound of structure,
Preferably, the general formula X-L-Y is the compound of structure shown in following formula (3-1),
It is highly preferred that general formula X-the L-Y is structure shown in the compound of structure and formula (3-1-2) shown in following formula (3-1-1)
Compound,
5. according to the fluorescence probe described in any one of claim 1-4, wherein the molecule of pharmaceutical activity containing flavonoids
Fluorescence probe is the compound of structure shown in the compound of structure and/or formula (3-1-2) shown in formula (3-1-1).
6. the preparation side of the fluorescence probe of the molecule of pharmaceutical activity containing flavonoids described in a kind of any one of claim 1-5
Method, which is characterized in that this approach includes the following steps,
1) compound of structure shown in formula (2) withAmide condensed reaction is carried out, is obtained
The midbody compound indicated to general formula L-Y, wherein n is the integer of 1-6;
2) make the midbody compound that general formula L-Y is indicated carry out Mannich with the compound of structure shown in formula (1) to react, obtain
The fluorescence probe for the molecule of pharmaceutical activity containing flavonoids that general formula X-L-Y is indicated.
7. according to the method described in claim 6, wherein, the condition of the amide condensed reaction includes:Reaction temperature is 15-80
DEG C, reaction time 10-72h.
8. according to the method described in claim 6, wherein, the condition of the Mannich reactions includes:Reaction temperature is 20-110
DEG C, reaction time 1-20h.
9. the fluorescence probe or claim 4- of the molecule of pharmaceutical activity containing flavonoids described in any one of claim 1-5
The fluorescence probe of the molecule of pharmaceutical activity containing flavonoids obtained by preparation method described in any one of 8 is preparing treatment tumour
Or the application in the drug of cancer.
10. application according to claim 9, wherein the tumour or cancer are lung cancer, cervical carcinoma, epidermis squamous carcinoma, mammary gland
Cancer or prostate cancer.
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