CN106632411B - A kind of antitumor fluorescent chemicals, preparation method and the usage - Google Patents
A kind of antitumor fluorescent chemicals, preparation method and the usage Download PDFInfo
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- CN106632411B CN106632411B CN201611202340.8A CN201611202340A CN106632411B CN 106632411 B CN106632411 B CN 106632411B CN 201611202340 A CN201611202340 A CN 201611202340A CN 106632411 B CN106632411 B CN 106632411B
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Abstract
The invention belongs to pharmaceutical technology field, more particularly to a kind of antitumor fluorescent chemicals, preparation method and the usage.The invention has the advantages that antitumor fluorescent chemicals provided by the invention carry fluorophor, fluorescent marker tracer can be carried out；And the compound is capable of the generation Bergman cyclizations under the acid condition of tumour cell of specificity, it can see from DNA experiments and cell experiment result, it has apparent chain splitting action to DNA and has apparent toxic effect to tumour cell, while realizing the high selectivity to tumour cell and normal cell.
Description
Technical field
The invention belongs to pharmaceutical technology field, more particularly to a kind of antitumor fluorescent chemicals, preparation method and the usage.
Background technology
Tumour is a kind of complex disease being related to unregulated cell growth, has become current threat human health, jeopardizes
One of major disease of life, more as one of the main reason for leading to death.For a long time, oncotherapy is all that medical field is ground
Study carefully the big project of absorbed one.The common medical procedure of tumour is operative treatment, biological therapy, radiotherapy and chemotherapy,
Middle chemotherapy is the major way of kinds of tumors.
More biologically active natural/synthesis class antibiotic compound, such as taxol, adriamycin etc. be always
The tumor therapeutic agent of mainstream, some have been widely used in clinic.However, most antitumor drugs also show that seriously
Side effect：They also have normal cell while killing tumour cell certain toxicity, such as：Thrombopenia and liver
Toxicity, long-time service can also make tumour cell generate drug resistance etc..Therefore, good anti-of new, specificity low with toxicity is developed
Tumour medicine has become an important directions in current organic and pharmaceutical chemistry.
Enediyne Antitumor Antibiotics are one kind of native tumor antibiotic, they have unique molecular structure, new
The mechanism of action and efficient antitumor activity of grain husk are one of the strongest antitumorigenic substances of activity having been found that so far,
With good development prospect, and have become chemistry, biology and field of medicaments primary study object.Many natural enediynes are swollen
Tumor antibiotic has very high cytotoxicity, is the apoptosis of inducible tumour cell in micro-molar concentration, such as
The half casualty-producing concentrations of Calicheamicin are horizontal in pg/ μ L, 1000 times stronger than adriamycin or more.Presently found enediyne
Neocarzinostatin has been used to clinical practice, primary treatment leukaemia, gastric cancer, cancer of pancreas etc. in class antibiotic.It is this kind of
Compound can generate double phenyl radicals by the way that Bergman Cyclization reactions occur, and directly act on and capturing with DNA
H atom on DNA molecular chain promotes apoptosis of tumor cells so as to cause the fracture of DNA chain from molecular level.However these alkene
Two acetylenic antitumor antibiotics still None- identified and distinguish normal cell and tumour cell, therefore have to normal cell certain
Toxic side effect.
Most of enediyne causes the condition of above-mentioned Bergman cyclisation or is that relatively high temperature heats so far,
It is ultraviolet lighting；Even if a small number of enediynes cause by metal ion or alkaline condition, but these conditions in human body all
Relatively it is difficult to realize.Human body maintains the physiology at cell or tissue in a partial neutral in normal metabolic process by acid-base balance
In environment；And canceration once occurs, tumour cell can be that the survival and reproduction of oneself create one by abnormal energetic supersession
It is unsuitable for the acidic micro-environment of normal cell existence.If using the acidic micro-environment in tumour cell, to cause enediyne
Bergman cyclizations, then enediyne can not only generate tumour cell toxicity, and also it is only micro- in the acidity of tumour cell
It reacts under environment, and Bergman cyclizations does not occur under the partial neutral environment of normal cell, can thus reach
The purpose of normal cell and tumour cell is correctly distinguished to enediyne, selective kill tumour cell and not to normal cell
It damages, this is that the novel highly selective antitumor enediyne of design has opened up new thinking.
The enediyne containing triethoxy group is designed and synthesized in patent CN201410211924.6, in acid item
DNA chain can be caused to be broken under part and can result in the apoptosis of tumour cell.But triethoxy group is to acid condition ratio
It is more sensitive, as long as opposite acid condition can initiation reaction, can not ensure under complicated cell micro-environment in human body
It reacts in tumour cell in due course and generates cytotoxicity；In addition, the compound is because no fluorescence can not also be marked
Tracer.
Invention content
The technical problem to be solved by the present invention is to：Overcome in the prior art antitumor drug to tumour cell and normal cell
Defect without selectivity provides a kind of antitumor fluorescent chemicals of selective kill tumour cell.
The technical solution adopted by the present invention to solve the technical problems is：A kind of antitumor fluorescent chemicals, structural formula is such as
Shown in lower：
The invention solves second technical problem be：Overcome in the prior art that antitumor drug is being to tumour cell and just
Normal cell does not have the deficiency of the defect of selectivity, provides the antitumor fluorescent chemicals of selective kill tumour cell
Preparation method.
Technical solution is used by the present invention solves second technical problem：A kind of preparation of antitumor fluorescent chemicals
Method includes the following steps：
By 3,4- bis- ((2- ethyoxyl -1,3- dioxolanes -2- bases) acetenyl) -1- (furans -2- ylmethyls) -1H- pyrroles
It coughs up -2,5- diketone to be dissolved in solvent, fluorescein -5- maleimides is added in reaction vessel in a nitrogen atmosphere, stir
After dissolving, sealing, which is reacted 0.5~10 hour, at 40~100 DEG C obtains compound b.
Preferably, the preparation method further includes following steps：
By iodo- 1H- pyrroles -2, the 5- diketone of 1- (furans -2- ylmethyls) -3,4-, iodate Asia ketone, POPd 1, triphenyl phosphorus
It is added sequentially in reaction vessel with cesium carbonate, weight steamed tetrahydrofuran and toluene, stirring and dissolving is added in a nitrogen atmosphere
Afterwards, it is eventually adding 2- ethyoxyls -2- acetenyls -1,3-dioxolane；System is stirred to react for 40~80 DEG C under nitrogen protection
0.5~10 hour, obtain compound a.
Preferably, the preparation method further includes following post-processing step：
Stop reaction postcooling to room temperature, with dichloromethane and saturated nacl aqueous solution extracting and washing, collected organic layer is used
Anhydrous magnesium sulfate is dried, and is concentrated filter vacuum after filtering, is obtained compound b.
Preferably, it is that prepare solvent described in the reaction of b be anhydrous DMF to raw material by a.
The invention solves third technical problem be：Overcome in the prior art that antitumor drug is being to tumour cell and just
Normal cell does not have the deficiency of the defect of selectivity, and the antitumor fluorescent chemicals for providing selective kill tumour cell exist
Prepare the application in antitumor drug.
Technical solution is used by the present invention solves third technical problem：Compound b is in the preparation of antitumor drugs
Application.
The invention has the advantages that antitumor fluorescence enediyne compound provided by the invention carries fluorophor, energy
Enough carry out fluorescent marker tracer；And the compound is capable of the generation Bergman rings under the acid condition of tumour cell of specificity
Change reaction, can see from cell experiment result, realize the high selectivity to tumour cell and normal cell.
Description of the drawings
Fig. 1-compound b cracks the chain of DNA at different pH.
Fig. 2-compound b is under various concentration, different time to the cytotoxicity of A549 tumour cells.
Fig. 3-compound b is under various concentration, different time to the cytotoxicity of L-02 normal cells.
Fig. 4-compound b is under various concentration to the cytotoxicity comparison diagram of A549 tumour cells and L-02 normal cells.
Fig. 5-compound b is imaged with the laser confocal microscope after mixing with cells.
Specific implementation mode
The synthesis of 1 compound e of embodiment
Succinic acid (0.1g, 0.87mmol), ethylene glycol (2.75g, 44.2mmol) and 3,3,3- triethoxies-are weighed respectively
1- propine (3.34g, 19.4mmol) is added in reaction bulb, and heating is allowed to return stirring and reacts 3 hours, stops reaction postcooling
To room temperature, with ethyl acetate and saturated sodium bicarbonate solution extracting and washing, collected organic layer is dry with anhydrous magnesium sulfate, filters,
Filtrate revolving is collected, obtained product obtains compound e, product warp after ethyl acetate/petroleum ether=1/10 chromatographs post separation
Hydrogen is composed and mass spectrum confirmation.
The synthesis of 2 compound d of embodiment
It weighs dibromomaleic acid acid anhydride (3g, 12mmol) to be added in round-bottomed flask, 80ml acetic acid is added, is stirred at room temperature molten
Solution.2- furylamines (1.37g, 14mmol) are added dropwise in flask, there is white cigarette generation immediately, are stirred at room temperature to white cigarette
It disappears, is added dropwise after white cigarette completely disappears after 2- furylamines and potassium iodide (3.7g, 22.4mmol) is added, reaction bulb is shifted
It is reacted 24 hours to being stirred at reflux in 120 DEG C of oil baths.Be cooled to room temperature after reaction, remove after acetic acid with ethyl acetate and
Saturated sodium bicarbonate solution is extracted, and collected organic layer is dried with anhydrous magnesium sulfate, and filtrate rotates after filtering, obtained solid
Compound d is obtained after column chromatography with ethyl acetate/petroleum ether=1/15, product is composed through hydrogen and mass spectrum confirmation.
The synthesis of 3 compound a of embodiment
By iodo- 1H- pyrroles -2, the 5- diketone (0.556g, 1.3mmol) of 1- (furans -2- ylmethyls) -3,4-, iodate Asia ketone
(100mg, 40mmol%), POPd 1 (121mg, 10mmol%), triphenyl phosphorus (68mg, 20mmol%) and cesium carbonate (1.3g,
3.99mmol) be added sequentially in the Schlenk bottles of 50ml, vacuum and exchange nitrogen three times, under a nitrogen be added weight it is steamed four
Hydrogen furans (4ml) and toluene (10ml) after stirring and dissolving, are eventually adding compound e (0.74g, 5.2mmol).In nitrogen protection
Lower 60 DEG C be stirred to react 4 hours after stop reaction, by column chromatography for separation (n-hexane/ethyl acetate=5/1), will be obtained after product
Hydrogen to compound a, compound a is composed and mass spectrometric data is as follows：
1H NMR(CDCl3, 400MHz, ppm)：δ 7.27 (s ,-O-CH=, 1H), 6.33-6.32 (d ,-CH=CH-, 1H),
6.30-6.29 (d ,-C=CH-, 1H), 4.71 (s ,-N-CH2-,2H),4.11-4.06(qq,-O-CH2-,8H),3.75-3.70
(q,CH3-CH2-,4H),1.33-1.26(t,-CH2-CH3,6H)。HRMS(ESI):m/z cald.For C23H23NO9Na(M+
Na)+:480.1271；found:480.1276.
The synthesis of 4 compound b of embodiment
Compound a (32mg, 0.069mmol) is dissolved in 5ml anhydrous DMFs in the sealed bottle of 10ml, vacuumizes and changes
Nitrogen three times after, weigh fluorescein -5- maleimides (33mg, 0.072mmol) be added in sealed bottle in a nitrogen atmosphere,
After stirring and dissolving, sealed bottle is placed in sealing reaction 8 hours at 80 DEG C.Stop reaction postcooling to room temperature, with dichloromethane and
Saturated nacl aqueous solution extracting and washing, collected organic layer are dried with anhydrous magnesium sulfate, concentrate filter vacuum after filtering, are changed
Object b is closed, the hydrogen spectrum and mass spectrometric data of compound b is as follows：
1H NMR(DMSO-d6,400MHz,ppm)：δ 6.43-8.22 (m ,-Ar-H, 9H), 6.05 (s ,-CH=CH, 1H),
6.17 (s ,-CH=CH, 1H), 4.63 (t ,-O-CH-, 1H), 4.11-4.06 (qq ,-O-CH2-,8H),4.02(s,-N-CH2-,
2H),3.75-3.70(q,CH3-CH2-,4H),2.86(d,-CH-CH-,1H),2.76(s,-CH-CH-,1H),1.33-1.26
(t,-CH2-CH3,6H)。HRMS(ESI):m/z cald.For C47H36N2O16Na(M+Na)+:907.1963；found:
907.1978。
5 compound b of embodiment tests the cracking of DNA
Compound b is made into the acetone soln of 50mM/L, 2 μ L is taken to be added in the DNA TE buffer solutions of 4 μ L, it is dense with difference
The para-methylbenzenepyridinsulfonate sulfonate (1mM/L, 10mM/L, 100mM/L, 1M/L) of degree adjusts different groups of other pH respectively in pH=respectively
3, pH=4, pH=5 and pH=6 or so.After being placed at 37 DEG C constant-temperature incubation 48 hours by different groups after mixing, take out molten
Liquid is tested into row agarose gel electrophoresis.
As shown in Figure 1, the experimental results showed that, under the group of pH=6 and pH=5, DNA has almost no change；In pH=4
When DNA variation just takes place, II forms of Form are converted by I forms of Form, illustrate DNA have occurred chain fracture；In pH=3
When, the form of DNA has more noticeable change, has the generation of II forms of more Form, illustrates that DNA molecular chain largely has occurred
Chain is broken.
The cytotoxicity experiment of 6 compound b of embodiment
Cytotoxic evaluation is carried out using MTT methods：
1. by exponential phase A549 tumour cells and L-02 normal cells be inoculated with after be placed on CO2Incubator
Middle adhere-wall culture；
2. compound b to be made into the acetone soln of 200mM/L, after being sterilized with membrane filtration, then distinguished with cell culture fluid
It is diluted to 100,50,25,12.5,6.25,3.1 μM/L；
3. the 200 μ L of compound b solution of each concentration is taken to be added separately to the tumour cell in culture and normal cell hole
In plate, 0.1% acetone of 200 μ L is added in blank group experiment；Orifice plate is placed on CO2Cultivated in incubator 24 hours, 48 hours with
And 72 hours；
4. being measured evaluation cell activity using MTT methods after culture.
The experimental results showed that adding the tumour cell of compound b, after having cultivated for 24 hours, significantly withering occurs in cell
It dies, with 48h, 72h of rising to of time, cell activity is substantially reduced, and illustrates that antitumoral compounds b shows tumour cell
Strong cytotoxicity, as shown in Figure 2.And on the contrary, compound b is obviously much smaller to the toxicity of normal cell, as shown in Figure 3.
The fluorescence of 7 compound b of embodiment
The process that compound b enters cell has been arrived using confocal laser scanning microscope, has been shown apparent yellowish green
Color fluorescence.As shown in Figure 5.
It is enlightenment with above-mentioned desirable embodiment according to the present invention, through the above description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property range is not limited to the contents of the specification, it is necessary to determine its technical scope according to right.
Claims (5)
1. a kind of antitumor fluorescent chemicals, which is characterized in that structural formula is as follows：
2. a kind of preparation method of antitumor fluorescent chemicals according to claim 1, it is characterised in that including walking as follows
Suddenly：
By 3,4- bis- ((2- ethyoxyl-1,3- dioxolanes-2- bases) acetenyl)-1- (furans-2- ylmethyls) pyrroles-2-1H-,
5- diketone is dissolved in solvent, fluorescein -5- maleimides is added in reaction vessel in a nitrogen atmosphere, stirring and dissolving
Afterwards, sealing reaction obtains compound b in 0.5~10 hour at 40~100 DEG C.
3. the preparation method of antitumor fluorescent chemicals as claimed in claim 2, it is characterised in that the preparation method is also wrapped
Include following post-processing step：
Stop reaction postcooling to room temperature, with dichloromethane and saturated nacl aqueous solution extracting and washing, collected organic layer is with anhydrous
Magnesium sulfate is dried, and is concentrated filter vacuum after filtering, is obtained compound b.
4. the preparation method of antitumor fluorescent chemicals as claimed in claim 2, it is characterised in that the solvent is anhydrous
DMF。
5. compound application in preparation of anti-tumor drugs according to claim 1.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980177A (en) * | 2014-05-20 | 2014-08-13 | 华东理工大学 | Acetylene compound as well as preparation method and application thereof |
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2016
- 2016-12-23 CN CN201611202340.8A patent/CN106632411B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103980177A (en) * | 2014-05-20 | 2014-08-13 | 华东理工大学 | Acetylene compound as well as preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| An acyclic enediyne with a furyl tethering group for efficient inhibition of tumor cell viability;Depeng Song,et al.,;《J. Mater. Chem. B》;20151231;第3卷;第8584-8588页 * |
| Maleimide-based acyclic enediyne for efficient DNA-cleavage and tumor cell suppression;Depeng Song,et al.,;《J. Mater. Chem. B》;20150217;第3卷;第3195-3200页 * |
| Multicolor Fluorescent Labeling of Cellulose Nanofibrils by Click Chemistry;Julien R. G. Navarro,et al.,;《Biomacromolecules》;20150316;第16卷;第1293-1300页 * |
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