CN108295258A - A kind of preparation method and applications of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation - Google Patents
A kind of preparation method and applications of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation Download PDFInfo
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Abstract
A kind of preparation method and applications of 10 hydroxycamptothecin/Porphyrin-Based Sensitizer compound formulation, it is related to a kind of preparation method and applications of Porphyrin-Based Sensitizer compound formulation.The present invention is to solve existing porphyrins photosensitizers for the ineffective technical problem of inhibition cancer cell survival rate.The present invention：One, dimerization L glutamic acid is prepared；Two, four poly- L glutamic acid are prepared；Three, six poly- L glutamic acid are prepared；Four, the bonded porphyrin dimer of six polyglutamic acids is prepared；Five, 10 hydroxycamptothecin/Porphyrin-Based Sensitizer compound formulation is prepared.10 hydroxycamptothecin/Porphyrin-Based Sensitizer compound formulation of the present invention is applied to inhibit the survival rate of cancer cell.10 hydroxycamptothecin/Porphyrin-Based Sensitizer compound formulation of the present invention can make the survival survival rate of cancer cell be reduced to 10% under conditions of illumination 40min.
Description
Technical field
The present invention relates to the preparation method of a kind of Porphyrin-Based Sensitizer and chemotherapeutics 10-hydroxycamptothecine compound formulation and
It is applied
Background technology
Porphyrins are that all organisms maintain substance necessary to bioactivity.It include a height in its molecule
The coplanar big ring of tetrapyrrole gripped altogether, at its big ring center possibly also with central metal atoms such as manganese or iron.The big ring of porphyrin
The presence of conjugated electron system makes their soret and Q band absorbing wavelength.Therefore, porphyrins cause countries in the world
The attention of researcher is to being applied to medical photosensitive agent.Due to the specific structural features of porphyrin itself, porphyrin tool
There is special optical performance, in addition porphyrin compound has special affinity with tumour cell, it can be a large amount of near cancer cell
Enrichment, can be used for the detection and treatment of cancer in medical research.Porphyrin is because porphyrin can be enriched with for cancer cell detection
The size and specific location of cancer cell can be accurately detected by the fluorescence signal of its stabilization on cancer cell.Why is porphyrin
Ability with kill cancer cell is because of it as a kind of photosensitizer drug, can be effective and selectivity thin in tumour
It is enriched on born of the same parents, when irradiating porphyrin enrichment region using the light of specific wavelength, porphyrin compound will produce active singlet
Oxygen, these singlet oxygens can with to the Interaction of substituents of oxidation-sensitive, cause in biomolecule tumour cell because of oxidation
Inactivation leads to death, to achieve the purpose that treatment.Photosensitive drug is irradiated by specific wavelength and the light of intensity, utilizes photosensitive medicine
The photodynamic reaction of object diagnoses tumour.The morning that can not be observed by detecting the detectable naked eyes of fluorescence that photosensitizer generates
Phase cancer or precancerous lesion, have the characteristics that noninvasive, quick, objective, recordable, repeatable, and can be indicated by characterized fluorescence
The infiltration degree of tumour carries out the level diagnosis of tumour, more thorough to the treatment of tumour.Photodynamic therapy has antitumor spectra
Extensively, wide indications, can be repeated several times treatment, can selective killing tumour cell, retain normal organ and tissue to large extent
Function.It is used in combination certain synergistic effect, photodynamic therapy and operation with chemotherapy and radiation and is used in combination, and can contract
The advantages that minor operation range and reduction postoperative recurrence.However at present about chemotherapeutics and photodynamic therapy combination for pernicious swollen
The preparation very little of tumor treatment.
Invention content
The present invention is to solve existing porphyrins photosensitizers for inhibiting cancer cell survival rate ineffective
Technical problem, and a kind of preparation method and applications of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation are provided.
The preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation of the present invention is to carry out according to the following steps
's：
One, the Pidolidone of 12.4g~14.7g and 100mL~150mL water are uniformly mixed, control mixing speed is
800r/min~1200r/min, stirring to Pidolidone are completely dissolved, and 16g~20g acetic anhydride is added, is then heated to 80 DEG C
~90 DEG C, continues to control mixing speed to be 800r/min~1200r/min, stir 30min~50min, be cooled to room temperature, control
Temperature is -20 DEG C, holding 12h~for 24 hours, and it then filters, filtering layer is washed using 0 DEG C~4 DEG C of ice water, after natural drying at 60 DEG C
Under the conditions of~80 DEG C, vacuum sublimation separation is carried out, two L-glutamic acid of White snowflake shape crystallized product being elevated and is not risen
The protected glutamic acid of product as off-white solid amino of China；
Two, two L-glutamic acids for obtaining 50mL~71mL thionyl chlorides and 120mg~128mg step 1 are in room temperature item
It is uniformly mixed under part, is heated to 76 DEG C~80 DEG C, keep the reaction condition the reaction was continued 2h~4h, then at reduced pressure conditions,
Distillation obtains reaction solution, control mixing speed is 800r/min~1200r/min, by reaction solution until no fraction steams
Be cooled to room temperature, add the Pidolidone of 29.4mg~35mg, and continue to be stirred to react 6h~8h, be added 100mL~
200mL redistilled waters filter controlled at 0 DEG C~4 DEG C, and 12g~16g acetic anhydride is added into filtrate, is heated to 80 DEG C
~90 DEG C, 30min~50min is continuously stirred, 8h~12h, filtering is then kept to subtract filtrate under the conditions of temperature is -20 DEG C
Distillation and concentration is pressed, it is then dry in the case where temperature is 60 DEG C~80 DEG C vacuum conditions, obtain four poly-L-glutamic acid of white powder product
Acid；
Three, four L-glutamic acids for obtaining 50mL~71mL thionyl chlorides and 110mg~128mg step 2 are in room temperature item
It is uniformly mixed under part, is heated to 70 DEG C~90 DEG C, kept 1.5h~2h, add 20mL~30mL anhydrous dimethyl sulphoxides,
Under reduced pressure, distills until no fraction steams, be then cooled to room temperature, add the L- paddy ammonia of 20.4mg~29.4mg
Then 100mL~200mL redistilled waters are added in acid, and the reaction was continued 6h~8h, filtered controlled at 0 DEG C~4 DEG C,
12mL~16mL acetic anhydride is added into filtrate, is heated to 80 DEG C~90 DEG C, control rotating speed for 800 turns/min~1200 turn/
Min continuously stirs 30min~50min, and 8h~12h, filtering is then kept to steam filtrate decompression under the conditions of temperature is -20 DEG C
Concentration is evaporated, it is then dry under 60 DEG C~80 DEG C vacuum conditions, obtain six L-glutamic acid of white powder product；
Four, six L-glutamic acids for obtaining 15mL~20mL thionyl chlorides and 77.5mg~80mg step 3 are in room temperature item
It is uniformly mixed under part, is heated to 80 DEG C~90 DEG C holding 2h~4h, the anhydrous n,N-Dimethylformamide of 5mL~10mL is added,
Under reduced pressure, unreacted thionyl chloride is removed, then stirring is cooled to room temperature, and 5- (the 4- hydroxyls of 150mL~200mL are added
Base phenyl) -10, the n,N-Dimethylformamide solution of 15,20- Triphenylporphyrins, wherein 5- (4- hydroxy phenyls) -10,15,
The quality of 20- Triphenylporphyrins is 124.4mg~157.8mg, the reaction was continued 4h~6h, and is heated to 90 DEG C~100 DEG C, is flowed back
1.5h, then stirring are cooled to room temperature, 30mL~50mL redistilled waters are added, and are heated to 90 DEG C~100 DEG C, reflux
1.5h, stirring are cooled to room temperature, and it is 6.5~7 to adjust pH value, at reduced pressure conditions, remove n,N-Dimethylformamide and water, very
Sky is dry, obtains aubergine powder, is then dissolved in aubergine powder in the mixed liquor of methanol and chloroform, is loaded into silica gel
Column carries out column chromatography and obtains the first chromatographic band first using chloroform as eluent, for the 5- (4- hydroxy phenyls)-not reacted completely
10,15,20- Triphenylporphyrins are collected the second chromatographic band, are then concentrated, vacuum then using methanol and chloroform as eluent
It is dry, obtain the bonded porphyrin dimer of six polyglutamic acids；
Five, the bonded porphyrin dimer of six polyglutamic acids prepared by 40mg~45mg step 4 is dissolved in 40mL~45mL's
In deionized water, the sodium bicarbonate aqueous solution of a concentration of 0.1g/mL is added dropwise under stirring conditions to the bonded porphin of six polyglutamic acids
The pH of quinoline dimer aqueous solution is 6.5~7；10-hydroxycamptothecine is dissolved in physiological saline, 10-hydroxycamptothecine is made
The saturated saline solution solution of 10-hydroxycamptothecine is slowly instilled six polyglutamic acids by funnel by saturated saline solution solution
In bonded porphyrin dimer aqueous solution, when solution colour from bluish violet become light red i.e. stop be added dropwise 10-hydroxycamptothecine it is molten
Solution then proceedes to stirring 2h~4h, the methanol of 40mL~45mL is added into solution, stand for 24 hours~48h, i.e., in physiological saline
Obtain 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation.
10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation of the present invention is applied to inhibit the survival rate of cancer cell.
The method comprises the steps of firstly, preparing a kind of dimerization porphyrin compounds of the bonded porphyrin of six polyglutamic acids, are a kind of water solubilitys
10-hydroxycamptothecine is embedded by good photosensitizer by a kind of simple method, to be prepared for a kind of 10- hydroxyls happiness
Alkali/Porphyrin-Based Sensitizer compound formulation is set, so as to which optical dynamic therapy and chemotherapy drugs in combination to be applied to the treatment of tumour.
In the step five of the present invention during normal saline solution of saturation 10-hydroxycamptothecine is added dropwise, pass through purple
Outer spectrophotometer monitoring, when originating dropwise addition, the Soret bands of porphyrin are constantly widened, after solution becomes light red, porphyrin
Soret bands no longer change.
10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation of the present invention can make cancer under conditions of illumination 40min
The survival survival rate of cell is reduced to 10%.
Description of the drawings
Fig. 1 is the reaction equation of the step three and step 4 of the present invention；
In the step of Fig. 2 is experiment one five, in the saturated saline solution solution that 10-hydroxycamptothecine is added dropwise to six polyglutamics
The ultraviolet light absorption curve of spectrum during in the bonded porphyrin dimer aqueous solution of acid；
Fig. 3 is the TEM figures for testing 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation that one prepares；
Fig. 4 is the graph of relation of the cell survival rate and testing time of testing one.
Specific implementation mode
Specific implementation mode one：Present embodiment is a kind of system of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation
Preparation Method specifically carries out according to the following steps：
One, the Pidolidone of 12.4g~14.7g and 100mL~150mL water are uniformly mixed, control mixing speed is
800r/min~1200r/min, stirring to Pidolidone are completely dissolved, and 16g~20g acetic anhydride is added, is then heated to 80 DEG C
~90 DEG C, continues to control mixing speed to be 800r/min~1200r/min, stir 30min~50min, be cooled to room temperature, control
Temperature is -20 DEG C, holding 12h~for 24 hours, and it then filters, filtering layer is washed using 0 DEG C~4 DEG C of ice water, after natural drying at 60 DEG C
Under the conditions of~80 DEG C, vacuum sublimation separation is carried out, two L-glutamic acid of White snowflake shape crystallized product being elevated and is not risen
The protected glutamic acid of product as off-white solid amino of China；
Two, two L-glutamic acids for obtaining 50mL~71mL thionyl chlorides and 120mg~128mg step 1 are in room temperature item
It is uniformly mixed under part, is heated to 76 DEG C~80 DEG C, keep the reaction condition the reaction was continued 2h~4h, then at reduced pressure conditions,
Distillation obtains reaction solution, control mixing speed is 800r/min~1200r/min, by reaction solution until no fraction steams
Be cooled to room temperature, add the Pidolidone of 29.4mg~35mg, and continue to be stirred to react 6h~8h, be added 100mL~
200mL redistilled waters filter controlled at 0 DEG C~4 DEG C, and 12g~16g acetic anhydride is added into filtrate, is heated to 80 DEG C
~90 DEG C, 30min~50min is continuously stirred, 8h~12h, filtering is then kept to subtract filtrate under the conditions of temperature is -20 DEG C
Distillation and concentration is pressed, it is then dry in the case where temperature is 60 DEG C~80 DEG C vacuum conditions, obtain four poly-L-glutamic acid of white powder product
Acid；
Three, four L-glutamic acids for obtaining 50mL~71mL thionyl chlorides and 110mg~128mg step 2 are in room temperature item
It is uniformly mixed under part, is heated to 70 DEG C~90 DEG C, kept 1.5h~2h, add 20mL~30mL anhydrous dimethyl sulphoxides,
Under reduced pressure, distills until no fraction steams, be then cooled to room temperature, add the L- paddy ammonia of 20.4mg~29.4mg
Then 100mL~200mL redistilled waters are added in acid, and the reaction was continued 6h~8h, filtered controlled at 0 DEG C~4 DEG C,
12mL~16mL acetic anhydride is added into filtrate, is heated to 80 DEG C~90 DEG C, control rotating speed for 800 turns/min~1200 turn/
Min continuously stirs 30min~50min, and 8h~12h, filtering is then kept to steam filtrate decompression under the conditions of temperature is -20 DEG C
Concentration is evaporated, it is then dry under 60 DEG C~80 DEG C vacuum conditions, obtain six L-glutamic acid of white powder product；
Four, six L-glutamic acids for obtaining 15mL~20mL thionyl chlorides and 77.5mg~80mg step 3 are in room temperature item
It is uniformly mixed under part, is heated to 80 DEG C~90 DEG C holding 2h~4h, the anhydrous n,N-Dimethylformamide of 5mL~10mL is added,
Under reduced pressure, unreacted thionyl chloride is removed, then stirring is cooled to room temperature, and 5- (the 4- hydroxyls of 150mL~200mL are added
Base phenyl) -10, the n,N-Dimethylformamide solution of 15,20- Triphenylporphyrins, wherein 5- (4- hydroxy phenyls) -10,15,
The quality of 20- Triphenylporphyrins is 124.4mg~157.8mg, the reaction was continued 4h~6h, and is heated to 90 DEG C~100 DEG C, is flowed back
1.5h, then stirring are cooled to room temperature, 30mL~50mL redistilled waters are added, and are heated to 90 DEG C~100 DEG C, reflux
1.5h, stirring are cooled to room temperature, and it is 6.5~7 to adjust pH value, at reduced pressure conditions, remove n,N-Dimethylformamide and water, very
Sky is dry, obtains aubergine powder, is then dissolved in aubergine powder in the mixed liquor of methanol and chloroform, is loaded into silica gel
Column carries out column chromatography and obtains the first chromatographic band first using chloroform as eluent, for the 5- (4- hydroxy phenyls)-not reacted completely
10,15,20- Triphenylporphyrins are collected the second chromatographic band, are then concentrated, vacuum then using methanol and chloroform as eluent
It is dry, obtain the bonded porphyrin dimer of six polyglutamic acids；
Five, the bonded porphyrin dimer of six polyglutamic acids prepared by 40mg~45mg step 4 is dissolved in 40mL~45mL's
In deionized water, the sodium bicarbonate aqueous solution of a concentration of 0.1g/mL is added dropwise under stirring conditions to the bonded porphin of six polyglutamic acids
The pH of quinoline dimer aqueous solution is 6.5~7；10-hydroxycamptothecine is dissolved in physiological saline, 10-hydroxycamptothecine is made
The saturated saline solution solution of 10-hydroxycamptothecine is slowly instilled six polyglutamic acids by funnel by saturated saline solution solution
In bonded porphyrin dimer aqueous solution, when solution colour from bluish violet become light red i.e. stop be added dropwise 10-hydroxycamptothecine it is molten
Solution then proceedes to stirring 2h~4h, the methanol of 40mL~45mL is added into solution, stand for 24 hours~48h, i.e., in physiological saline
Obtain 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation.
Specific implementation mode two：The present embodiment is different from the first embodiment in that：By the L- of 13g in step 1
Glutamic acid and 120mL water are uniformly mixed, and control mixing speed is 1000r/min, and stirring to Pidolidone is completely dissolved.Other
It is same as the specific embodiment one.
Specific implementation mode three：The present embodiment is different from the first and the second embodiment in that：By 61mL in step 2
Two L-glutamic acids that thionyl chloride and 125mg step 1 obtain are uniformly mixed at ambient temperature, are heated to 80 DEG C, and keeping should
Then at reduced pressure conditions reaction condition the reaction was continued 3h distills until no fraction steams, obtains reaction solution.Other with
Specific implementation mode one or two is identical.
Specific implementation mode four：Unlike one of present embodiment and specific implementation mode one to three：It will in step 3
Four L-glutamic acids that 61mL thionyl chlorides and 125mg step 2 obtain are uniformly mixed at ambient temperature, are heated to 80 DEG C, are protected
2h is held, 30mL anhydrous dimethyl sulphoxides are added, at reduced pressure conditions, distills until no fraction steams, is then cooled to
Room temperature.Other are identical as one of specific implementation mode one to three.
Specific implementation mode five：Unlike one of present embodiment and specific implementation mode one to four：It will in step 4
Six L-glutamic acids that 18mL thionyl chlorides and 80mg step 3 obtain are uniformly mixed at ambient temperature, are heated to 805 DEG C of guarantors
3h is held, the anhydrous n,N-Dimethylformamide of 7mL is added and removes unreacted thionyl chloride at reduced pressure conditions.Other with it is specific
One of embodiment one to four is identical.
Specific implementation mode six：Unlike one of present embodiment and specific implementation mode one to five：10- hydroxy-camptothecins
Alkali/Porphyrin-Based Sensitizer compound formulation is applied to inhibit the survival rate of cancer cell.One of other and specific implementation mode one to five
It is identical.
The present invention is verified with following tests：
Experiment one：This experiment is a kind of preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation, specifically
It carries out according to the following steps：
One, the Pidolidone of 14g and 130mL water are uniformly mixed, control mixing speed is 1000r/min, and stirring is extremely
Pidolidone is completely dissolved, and 18g acetic anhydride is added, is then heated to 90 DEG C, is continued to control mixing speed to be 1000r/min, be stirred
40min is mixed, room temperature is cooled to, controlled at -20 DEG C, 20h is kept, then filters, filter is washed using 0 DEG C~4 DEG C of ice water
Layer carries out vacuum sublimation separation, the White snowflake shape crystallized product dimerization L- being elevated after natural drying under the conditions of 70 DEG C
Glutamic acid and the protected glutamic acid of product as off-white solid amino not distilled；
Two, two L-glutamic acids that 61mL thionyl chlorides and 125mg step 1 obtain are uniformly mixed at ambient temperature,
80 DEG C are heated to, keeps the reaction condition the reaction was continued then at reduced pressure conditions 3h distills until no fraction steams,
Reaction solution is obtained, control mixing speed is 1000r/min, and reaction solution is cooled to room temperature, adds the Pidolidone of 32mg, and
And continue to be stirred to react 7h, 150mL redistilled waters are added, are filtered controlled at 2 DEG C, 14g acetic anhydride is added into filtrate,
85 DEG C are heated to, 40min is continuously stirred, then keeps 10h under the conditions of temperature is -20 DEG C, filters, filtrate decompression is distilled dense
Contracting, it is then dry in the case where temperature is 70 DEG C of vacuum conditions, obtain four L-glutamic acid of white powder product；
Three, four L-glutamic acids that 61mL thionyl chlorides and 125mg step 2 obtain are uniformly mixed at ambient temperature,
Be heated to 80 DEG C, keep 2h, add 25mL anhydrous dimethyl sulphoxides, at reduced pressure conditions, distillation until no fraction steam for
Only, it is then cooled to room temperature, adds the Pidolidone of 25.4mg, and the reaction was continued 7h, 150mL second distillations are then added
Water filters controlled at 2 DEG C, and 15mL acetic anhydride is added into filtrate, is heated to 85 DEG C, and control rotating speed is 1000 turns/min,
40min is continuously stirred, then keeps 10h under the conditions of temperature is -20 DEG C, is filtered, by filtrate decompression distillation and concentration, then 70
It is dry under DEG C vacuum condition, obtain six L-glutamic acid of white powder product；
Four, six L-glutamic acids that 20mL thionyl chlorides and 80mg step 3 obtain are uniformly mixed at ambient temperature,
90 DEG C of holding 3h are heated to, the anhydrous n,N-Dimethylformamide of 8mL is added, at reduced pressure conditions, it is sub- to remove unreacted dichloro
Sulfone, then stirring are cooled to room temperature, the 5- (4- hydroxy phenyls) -10 of 180mL, the N of 15,20- Triphenylporphyrins, N- diformazans are added
The quality of base formamide solution, wherein 5- (4- hydroxy phenyls) -10,15,20- Triphenylporphyrins is 147.8mg, and the reaction was continued
5h, and 95 DEG C are heated to, flow back 1.5h, and then stirring is cooled to room temperature, and 40mL redistilled waters are added, are heated to 95 DEG C,
Flow back 1.5h, and stirring is cooled to room temperature, and it is 6.5~7 to adjust pH value, at reduced pressure conditions, remove n,N-Dimethylformamide and
Water, vacuum drying, obtains aubergine powder, is then dissolved in aubergine powder in the mixed liquor of methanol and chloroform, is loaded into
Silicagel column carries out column chromatography and obtains the first chromatographic band first using chloroform as eluent, for 5- (the 4- hydroxy benzenes not reacted completely
Base) -10,15,20- Triphenylporphyrins are collected the second chromatographic band, are then concentrated then using methanol and chloroform as eluent,
Vacuum drying, obtains the bonded porphyrin dimer of six polyglutamic acids；
Five, the bonded porphyrin dimer of six polyglutamic acids prepared by 40mg step 4 is dissolved in the deionized water of 40mL,
The sodium bicarbonate aqueous solution that a concentration of 0.1g/mL is added dropwise under stirring conditions is water-soluble to the bonded porphyrin dimer of six polyglutamic acids
The pH of liquid is 6.5~7；10-hydroxycamptothecine is dissolved in the saturated saline solution that 10-hydroxycamptothecine is made in physiological saline
The saturated saline solution solution of 10-hydroxycamptothecine is slowly instilled the bonded porphyrin dimerization of six polyglutamic acids by solution by funnel
In object aqueous solution, when solution colour from bluish violet become light red i.e. stop be added dropwise 10-hydroxycamptothecine be dissolved in physiological saline,
Stirring 3h is then proceeded to, the methanol of 40mL is added into solution, stands 36h, that is, it is photosensitive to obtain 10-hydroxycamptothecine/porphyrin
Agent compound formulation.
In five this experiment the step of, in the saturated saline solution solution that 10-hydroxycamptothecine is added dropwise to six polyglutamic acids
It during in bonded porphyrin dimer aqueous solution, is monitored using ultraviolet specrophotometer, such as the ultraviolet light absorption spectrum of Fig. 2
Shown in curve, arrow direction indicates that the process that the saturated saline solution solution of 10-hydroxycamptothecine is added dropwise, simple porphyrin are molten
Liquid, a strong porphyrin Soret absorption band appears in 420nm, however as being continuously added for 10-hydroxycamptothecine, the absorption
Band is constantly widened, and illustrates 10 hydroxycamptothecin by hydrogen bond insertion porphyrin dimer, when being added drop-wise to ultra-violet absorption spectrum bands of a spectrum
Do not changing, is stopping that 10-hydroxycamptothecine is added dropwise, illustrate that 10-hydroxycamptothecine has reached maximum concentration.
10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation of this experiment preparation is measured by light scattering particle size instrument
Grain size, the results show that the grain size of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation is about 166nm, and it is close single
Dispersity, dispersion degree 0.196.
Further, Fig. 3 is the TEM figures of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation prepared by this experiment, knot
Fruit shows that the compound formulation is spherical morphology.
Experiment two：Thiophene indigo plant colorimetric method (mtt assay) is selected, is divided into five groups：First group is control group：Illumination physiological saline group；
The bonded porphyrin dimer aqueous solution of six polyglutamic acids prepared by second group of one step 4 of experiment for 50 mcg/mls carries out half-light
Test；Third group is simple light group, does not add any drug；Prepared by the 4th group of one step 4 of experiment for 50 mcg/mls
The bonded porphyrin dimer aqueous solution of six polyglutamic acids carries out illumination test；Prepared by the 5th group of experiment one for 50 mcg/mls
The aqueous solution light group of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation.
Breast cancer cell is prepared into a concentration of 1 × 105~5 × 105The cell suspension of a/milliliter takes 0.1 milliliter of addition
96 orifice plates, while above-mentioned five groups of tested material is added, every group sets three parallel holes, is placed in 37 DEG C, contains carbon dioxide volume point
After number is culture 48h in the incubator of 5% air atmosphere, 10 microlitres of MTT is added per hole, continues to incubate 4h, light group is being kept away
Each hole cell of laser irradiation for being 532 nanometers with wavelength under the conditions of light, 2.4 joule/square centimeter of laser energy density of irradiation.
Non- irradiation hole is covered when irradiation so as not to light scattering influence experimental result.Then per hole, the mass fraction of 0.1 milliliter of addition is
The n,N-Dimethylformamide that 20% lauryl sodium sulfate and 0.1 milliliter of mass fraction are 50%, makes the crystallization of generation
After being completely dissolved, the OD value on the upside of automatic enzyme mark analyzer calculates cell survival rate：
Cell survival rate=(control group OD- test group OD/ test group OD) × 100%
Fig. 4 is the graph of relation of the cell survival rate and testing time of this experiment, the results show that in 50 mcg/mls
The six polyglutamic acids bonded porphyrin dimer aqueous solution for preparing of one step 4 of experiment when carrying out half-light test, six polyglutamic acid keys
Join lethality very little of the porphyrin dimer aqueous solution to breast cancer cell；
Only use up the irradiation non-dosing object of cancer cell, the lethality very little of breast cancer tumor cells, only drug and light group
Close just has strong lethal effect to breast cancer cell.
To the lethal effect of tumour cell, 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation is in identical illumination condition
Under, the bonded porphyrin dimer of six polyglutamic acids is substantially better than to the killing ability of tumour cell, this illustrates that compound formulation has
Synergistic effect；Not only porphyrin dimer photosensitizer has fragmentation effect, but also the antitumor effect of 10-hydroxycamptothecine to tumour cell
It should still remain.
The 10-hydroxycamptothecine of this experiment/Porphyrin-Based Sensitizer compound formulation can make cancer under conditions of illumination 40min
The survival rate of cell is reduced to 10%, and simple porphyrin dimer photosensitizer illumination after forty minutes, cell survival rate exists
30% or more.
Claims (6)
1. a kind of preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation, it is characterised in that 10- hydroxy-camptothecins
The preparation method of alkali/Porphyrin-Based Sensitizer compound formulation carries out according to the following steps：
One, the Pidolidone of 12.4g~14.7g and 100mL~150mL water are uniformly mixed, control mixing speed is 800r/
Min~1200r/min, stirring to Pidolidone are completely dissolved, and 16g~20g acetic anhydride is added, is then heated to 80 DEG C~90
DEG C, continue to control mixing speed to be 800r/min~1200r/min, stirs 30min~50min, be cooled to room temperature, control temperature
It is -20 DEG C, holding 12h~for 24 hours, it then filters, filtering layer is washed using 0 DEG C~4 DEG C of ice water, after natural drying 60 DEG C~80
Under the conditions of DEG C, vacuum sublimation separation is carried out, two L-glutamic acid of White snowflake shape crystallized product that is elevated and do not distil
The protected glutamic acid of product as off-white solid amino；
Two, two L-glutamic acids for obtaining 50mL~71mL thionyl chlorides and 120mg~128mg step 1 are at ambient temperature
It is uniformly mixed, is heated to 76 DEG C~80 DEG C, keep the reaction condition the reaction was continued 2h~4h, then at reduced pressure conditions, distillation
Until no fraction steams, reaction solution is obtained, control mixing speed is 800r/min~1200r/min, and reaction solution is cooled down
To room temperature, the Pidolidone of 29.4mg~35.0mg is added, and continues to be stirred to react 6h~8h, 100mL~200mL is added
Redistilled water filters controlled at 0 DEG C~4 DEG C, and 12g~16g acetic anhydride is added into filtrate, is heated to 80 DEG C~90
DEG C, 30min~50min is continuously stirred, 8h~12h, filtering is then kept to steam filtrate decompression under the conditions of temperature is -20 DEG C
Concentration is evaporated, it is then dry in the case where temperature is 60 DEG C~80 DEG C vacuum conditions, obtain four L-glutamic acid of white powder product；
Three, four L-glutamic acids for obtaining 50mL~71mL thionyl chlorides and 110mg~128mg step 2 are at ambient temperature
It is uniformly mixed, is heated to 70 DEG C~90 DEG C, keep 1.5h~2h, add 20mL~30mL anhydrous dimethyl sulphoxides, depressurizing
Under the conditions of, it distills until no fraction steams, is then cooled to room temperature, adds the Pidolidone of 20.4mg~29.4mg,
And then 100mL~200mL redistilled waters are added in the reaction was continued 6h~8h, filtered controlled at 0 DEG C~4 DEG C, to filter
12mL~16mL acetic anhydride is added in liquid, is heated to 80 DEG C~90 DEG C, control rotating speed is 800 turns/min~1200 turn/min, even
Continuous stirring 30min~50min, then keeps 8h~12h, filtering to distill filtrate decompression dense under the conditions of temperature is -20 DEG C
Contracting, it is then dry under 60 DEG C~80 DEG C vacuum conditions, obtain six L-glutamic acid of white powder product；
Four, six L-glutamic acids for obtaining 15mL~20mL thionyl chlorides and 77.5mg~80mg step 3 are at ambient temperature
It is uniformly mixed, is heated to 80 DEG C~90 DEG C holding 2h~4h, the anhydrous n,N-Dimethylformamide of 5mL~10mL is added, is depressurizing
Under the conditions of, unreacted thionyl chloride is removed, then stirring is cooled to room temperature, and 5- (the 4- hydroxy benzenes of 150mL~200mL is added
Base) -10, the n,N-Dimethylformamide solution of 15,20- Triphenylporphyrins, wherein 5- (4- hydroxy phenyls) -10,15,20- tri-
The quality of phenyl porphyrin is 124.4mg~157.8mg, the reaction was continued 4h~6h, and is heated to 90 DEG C~100 DEG C, and flow back 1.5h,
Then stirring is cooled to room temperature, and 30mL~50mL redistilled waters are added, and is heated to 90 DEG C~100 DEG C, and flow back 1.5h, stirring
It is cooled to room temperature, it is 6.5~7 to adjust pH value, at reduced pressure conditions, removes n,N-Dimethylformamide and water, vacuum drying obtains
To aubergine powder, then aubergine powder is dissolved in the mixed liquor of methanol and chloroform, is loaded into silicagel column, carries out column layer
Analysis, first using chloroform as eluent, obtains the first chromatographic band, for the 5- (4- hydroxy phenyls) -10,15,20- tri- not reacted completely
Phenyl porphyrin is collected the second chromatographic band, is then concentrated then using methanol and chloroform as eluent, is dried in vacuo, obtains six
The bonded porphyrin dimer of polyglutamic acid；
Five, by 40mg~45mg step 4 prepare the bonded porphyrin dimer of six polyglutamic acids be dissolved in 40mL~45mL go from
In sub- water, the sodium bicarbonate aqueous solution of a concentration of 0.1g/mL is added dropwise under stirring conditions to the bonded porphyrin of six polyglutamic acids two
The pH of polymers aqueous solution is 6.5~7；10-hydroxycamptothecine is dissolved in the saturation that 10-hydroxycamptothecine is made in physiological saline
It is bonded slowly to be instilled by funnel six polyglutamic acids by normal saline solution for the saturated saline solution solution of 10-hydroxycamptothecine
In porphyrin dimer aqueous solution, when solution colour from bluish violet become light red i.e. stop be added dropwise 10-hydroxycamptothecine be dissolved in
Physiological saline then proceedes to stirring 2h~4h, the methanol of 40mL~45mL is added into solution, stands for 24 hours~48h, that is, obtain
10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation.
2. a kind of preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation according to claim 1,
It is characterized in that Pidolidone and the 120mL water in step 1 by 13g are uniformly mixed, control mixing speed is 1000r/min, is stirred
It mixes to Pidolidone and is completely dissolved.
3. a kind of preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation according to claim 1,
It is characterized in that mixing two L-glutamic acids that 61mL thionyl chlorides and 125mg step 1 obtain at ambient temperature in step 2
Uniformly, 80 DEG C are heated to, keeps the reaction condition the reaction was continued 3h, then at reduced pressure conditions, distillation is steamed until no fraction
Until, obtain reaction solution.
4. a kind of preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation according to claim 1,
It is characterized in that mixing four L-glutamic acids that 61mL thionyl chlorides and 125mg step 2 obtain at ambient temperature in step 3
Uniformly, 80 DEG C are heated to, 2h is kept, adds 30mL anhydrous dimethyl sulphoxides, at reduced pressure conditions, distillation is steamed until no fraction
Until going out, it is then cooled to room temperature.
5. a kind of preparation method of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation according to claim 1,
It is characterized in that mixing six L-glutamic acids that 18mL thionyl chlorides and 80mg step 3 obtain at ambient temperature in step 4
Uniformly, 805 DEG C of holding 3h are heated to, the anhydrous n,N-Dimethylformamide of 7mL is added, at reduced pressure conditions, are removed unreacted
Thionyl chloride.
6. a kind of application of 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation as described in claim 1, feature exist
It is applied to inhibit the survival rate of cancer cell in 10-hydroxycamptothecine/Porphyrin-Based Sensitizer compound formulation.
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