CN111606919B - Solvate of carboxyfluorescein succinimidyl ester and preparation method thereof - Google Patents
Solvate of carboxyfluorescein succinimidyl ester and preparation method thereof Download PDFInfo
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- CN111606919B CN111606919B CN202010442336.9A CN202010442336A CN111606919B CN 111606919 B CN111606919 B CN 111606919B CN 202010442336 A CN202010442336 A CN 202010442336A CN 111606919 B CN111606919 B CN 111606919B
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- carboxyfluorescein
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- 239000012453 solvate Substances 0.000 title claims abstract description 29
- VDABVNMGKGUPEY-UHFFFAOYSA-N 6-carboxyfluorescein succinimidyl ester Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=C2)OC(=O)C1=CC=C2C(=O)ON1C(=O)CCC1=O VDABVNMGKGUPEY-UHFFFAOYSA-N 0.000 title claims description 12
- 238000002360 preparation method Methods 0.000 title abstract description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 123
- -1 carboxyfluorescein succinimide ester Chemical class 0.000 claims abstract description 47
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 46
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229960002317 succinimide Drugs 0.000 claims abstract description 40
- 239000002904 solvent Substances 0.000 claims abstract description 14
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 38
- 238000006243 chemical reaction Methods 0.000 claims description 27
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 25
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 22
- 239000007787 solid Substances 0.000 claims description 20
- 239000003480 eluent Substances 0.000 claims description 19
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 19
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 19
- 238000006482 condensation reaction Methods 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 14
- 238000001704 evaporation Methods 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 14
- 239000000741 silica gel Substances 0.000 claims description 14
- 229910002027 silica gel Inorganic materials 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 12
- 239000000706 filtrate Substances 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 238000009833 condensation Methods 0.000 claims description 9
- 230000005494 condensation Effects 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 238000004537 pulping Methods 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 claims description 3
- 239000007810 chemical reaction solvent Substances 0.000 claims description 3
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 22
- 239000003814 drug Substances 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 239000012535 impurity Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 238000012544 monitoring process Methods 0.000 description 8
- 239000000843 powder Substances 0.000 description 7
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 238000007614 solvation Methods 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012065 filter cake Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 238000011097 chromatography purification Methods 0.000 description 3
- 238000007086 side reaction Methods 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000012921 fluorescence analysis Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000007922 dissolution test Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a carboxyfluorescein succinimide ester solvate and a preparation method thereof. The solvent in the solvate is ethyl acetate or acetone. For fluorescein and its derivative carboxyfluorescein succinimide ester, the compound is prepared into a solvate, and especially ethyl acetate or acetone is selected as a solvent, so that the dispersibility of the solvate in water can be effectively improved, and the application effect of the compound in the aspect of drug tracing can be improved.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a carboxyfluorescein succinimide ester solvate and a preparation method thereof.
Background
Fluorescein and its derivative succinimidyl ester are widely used in fluorescence analysis, tracing dye, pH indicator and the derivative for synthesizing carboxyfluorescein, etc., are intracellular pH probes and are widely used in the medical field.
However, the existing fluorescein derivative succinimide ester has poor dispersibility in water and low solubility, so that the application of the fluorescein derivative serving as a pharmaceutic adjuvant in the aspect of drug tracing is restricted, and particularly the fluorescein derivative serving as a pharmaceutic adjuvant in the aspect of drug tracing is limited.
Based on the above current situation, there is an urgent need to improve the dissolution of the fluorescein derivative succinimidyl ester in water, so as to overcome the disadvantages of the prior art and prepare a high-quality fluorescein derivative succinimidyl ester product.
Disclosure of Invention
Aiming at the problem that the fluorescein derivative succinimide ester in the prior art has low solubility in water, the invention firstly provides a solvate of carboxyl fluorescein succinimide ester, and the solvent in the solvate is ethyl acetate or acetone.
There are many ways to improve the solubility of carboxyfluorescein succinimidyl ester in water, such as adding dissolution promoter. However, carboxyfluorescein succinimidyl ester is generally applied to the fields of fluorescence analysis and tracing, and the carboxyfluorescein succinimidyl ester is combined with an active ingredient to act independently in the using process, so that after being dissolved in water, a dissolving promoter needs to be effectively separated from the carboxyfluorescein succinimidyl ester, and the dissolving promoter does not influence the combination of the carboxyfluorescein succinimidyl ester and the active ingredient. The dissolution promoter has the problems that the residue is difficult to remove and the safety of the product is influenced. If the compound is prepared into the solvate, various factors such as the relative polarity of the solvent, the fluorescein succinimide ester and water, the separation condition of the solvent and the fluorescein succinimide ester in the solvent, the feasibility of the preparation of the chemical compound, the safety of the solvent in a human body, the characteristic of low boiling point and the like need to be considered, and the inventors surprisingly found that the solvate prepared by taking ethyl acetate or acetone as the solvent can meet the requirements of improving the water solubility of the fluorescein succinimide ester and simultaneously meeting the aspects, wherein the effect of the ethyl acetate is more prominent.
Preferably, 0.5 to 1 molecule of ethyl acetate is added to each molecule of carboxyfluorescein succinimide ester.
In a preferred embodiment, the carboxyfluorescein succinimide ester is 5(6) -carboxyfluorescein succinimide ester.
The invention also improves the preparation method of carboxyl fluorescein succinimide ester, which comprises the following steps:
performing condensation reaction by using carboxyl fluorescein and N-hydroxysuccinimide as raw materials to obtain a carboxyl fluorescein succinimide ester primary product; in the process of condensation reaction, N-dicyclohexyl carbodiimide or 1-ethyl-3 (3-dimethylpropylamine) carbodiimide hydrochloride is used as a condensing agent; the temperature of the condensation reaction is controlled to be 20-30 ℃.
The method can effectively control the side reaction by selecting a proper condensation reagent and controlling the reaction temperature to be 20-30 ℃, the obtained solvate prepared from the carboxyfluorescein succinimide ester has good dispersibility in water, and the reaction process of the method is easy to control.
In a preferable operation mode, the carboxyfluorescein is 5(6) -carboxyfluorescein, and the mass ratio of the 5-carboxyfluorescein to the 6-carboxyfluorescein in the 5(6) -carboxyfluorescein is 1: 1-5.
As a preferred mode of operation, the condensing agent is added in portions during the reaction.
In a preferred mode of operation, the condensation reaction uses tetrahydrofuran as a reaction solvent.
As a preferable operation mode, the method further comprises the step of separating and purifying the carboxyfluorescein succinimide ester primary product by chromatography after the condensation is completed, and the method comprises the following steps:
1) purifying the solution obtained after the condensation reaction is finished, mixing the solution with silica gel, and evaporating the mixture to dryness under reduced pressure to obtain silica gel to be treated;
2) and eluting the silica gel to be treated by using a mixed solution of ethyl acetate and normal hexane as an eluent, and purifying the eluent to obtain the carboxyfluorescein succinimide ester.
As a preferred mode of operation, the specific steps of purification of the solution obtained after the end of the condensation reaction are: filtering the obtained solution, evaporating the filtrate at 30-50 ℃ under reduced pressure to remove the solvent, adding acetone into the residue, stirring at 15-40 ℃ for 0.5-1 h, and filtering out insoluble substances.
As a preferred operation mode, the specific steps of purifying the eluent are as follows: concentrating the eluent under reduced pressure at 20-50 ℃ until no liquid drops, adding methyl tert-butyl ether into the residual solid, pulping at 20-40 ℃ until the solid is uniformly dispersed, and distilling under reduced pressure at 20-50 ℃ until the solid is dried.
Another object of the present invention is to protect the process for the preparation of the solvate according to the invention, comprising the following steps: mixing carboxyfluorescein succinimidyl ester with ethyl acetate or acetone, and volatilizing ethyl acetate or acetone.
In a preferred mode of operation, the ethyl acetate or acetone is volatilized and dried under vacuum at 20 to 50 ℃.
The invention also protects the application of the solvate in the field of medicines.
Detailed Description
The present invention will be described in detail by the following additional technical features.
The invention firstly provides a solvate of carboxyfluorescein succinimide ester, wherein a solvent in the solvate is ethyl acetate or acetone.
According to some preferred embodiments, ethyl acetate is chosen as solvent. Both ethyl acetate and acetone can be used as solvents, but ethyl acetate is more effective and safer.
According to some preferred embodiments, the carboxyfluorescein succinimide ester corresponds to 0.5-1 molecule of ethyl acetate per molecule. The above amount is more favorable for improving the solubility and the dispersibility of the carboxyfluorescein succinimide ester.
According to some preferred embodiments, the carboxyfluorescein succinimide ester is 5(6) -carboxyfluorescein succinimide ester. 5(6) -carboxyl fluorescein succinimidyl ester has good associativity with ethyl acetate, low production cost and excellent effect in the aspect of medicine tracing, and is favorable for large-scale popularization and application.
According to some preferred embodiments, the carboxyfluorescein succinimide ester is prepared by a process comprising the steps of:
performing condensation reaction by using carboxyl fluorescein and N-hydroxysuccinimide as raw materials to obtain a carboxyl fluorescein succinimide ester primary product;
in the process of condensation reaction, N-dicyclohexyl carbodiimide or 1-ethyl-3 (3-dimethylpropylamine) carbodiimide hydrochloride is used as a condensing agent; controlling the temperature of the condensation reaction to be 20-30 ℃;
the method can effectively control the side reaction by selecting a proper condensation reagent and controlling the reaction temperature to be 20-30 ℃, improves the binding capacity of the carboxyfluorescein succinimide ester and the ethyl acetate and improves the dispersibility in water, and the reaction process is easy to control.
Specifically, the condensing agent is added in batches in the reaction process to control the reaction, so that the reaction temperature is more easily maintained at 20-30 ℃.
Specifically, the carboxyfluorescein is 5(6) -carboxyfluorescein, and the mass ratio of the 5-carboxyfluorescein to the 6-carboxyfluorescein in the 5(6) -carboxyfluorescein is 1: 1-5; the 5(6) -carboxyl fluorescein succinimidyl ester prepared by the proportion has the advantages of optimal quality and yield.
Specifically, Tetrahydrofuran (THF) is used as a reaction solvent during the condensation reaction.
According to some preferred embodiments, 5(6) -carboxyfluorescein: NHS (N-hydroxysuccinimide): the feeding ratio of DCC (N, N-dicyclohexylcarbodiimide) is 1: 0.2-0.6: 0.5-1.0. The 5(6) -carboxyl fluorescein succinimidyl ester prepared by the proportion has the advantages of optimal quality and yield.
According to some preferred embodiments, the condensation is further performed by separating and purifying the carboxyfluorescein succinimidyl ester crude product by chromatography, comprising the following steps:
1) purifying the solution obtained after the condensation reaction is finished, mixing the solution with silica gel, and evaporating the mixture to dryness under reduced pressure to obtain silica gel to be treated;
2) and eluting the silica gel to be treated by using a mixed solution of ethyl acetate and normal hexane as an eluent, and purifying the eluent to obtain the carboxyfluorescein succinimide ester.
According to some preferred embodiments, the ratio of Ethyl Acetate (EA) to n-ethane is 1:1 to 5. The 5(6) -carboxyl fluorescein succinimidyl ester prepared by the proportion has the advantages of optimal quality and yield.
According to some preferred embodiments, the specific steps of purification of the solution obtained after the end of the condensation reaction are: filtering the obtained solution, evaporating the filtrate at 30-50 ℃ under reduced pressure to remove the solvent, adding acetone into the residue, stirring at 15-40 ℃ for 0.5-1 h, and filtering out insoluble substances.
According to some preferred embodiments, the specific steps of purifying the eluate are: concentrating the eluent under reduced pressure at 20-50 ℃ until no liquid drops, adding methyl tert-butyl ether into the residual solid, pulping at 20-40 ℃ until the solid is uniformly dispersed, and then distilling under reduced pressure at 20-50 ℃ until the solid is dried to obtain a carboxyfluorescein succinimide ester crude product.
Specifically, the chromatography comprises the following steps:
1) after the reaction is finished, filtering the reaction solution, leaching a filter cake with THF, draining, evaporating THF from the filtrate under reduced pressure in a water bath at 30-50 ℃, adding acetone into the residue, stirring at 15-40 ℃, filtering out insoluble substances, adding 100-200 meshes of silica gel into the filtrate, and evaporating to dryness under reduced pressure at 30-50 ℃ to obtain silica gel to be treated;
2) performing column chromatography separation and purification, wherein the eluent comprises ethyl acetate and n-hexane
TLC monitoring of the effluent, starting collection when TLC monitoring of the effluent shows a clear target product spot. Collecting eluent, concentrating under reduced pressure at 20-50 ℃ (vacuum degree is less than or equal to-0.09 MPa) until no liquid drops drop, adding methyl tert-butyl ether into the residue, pulping at 20-40 ℃ until the solid is uniformly dispersed, and distilling under reduced pressure at 20-50 ℃ until the solid is dried to obtain a 5(6) -carboxyl fluorescein succinimidyl ester crude product.
The invention also provides a preparation method of the solvate, which comprises the steps of dissolving carboxyl fluorescein succinimide ester in ethyl acetate or acetone, and volatilizing the ethyl acetate or the acetone.
According to some preferred embodiments, the ethyl acetate or acetone is volatilized and dried under vacuum at 20-50 ℃, and the drying temperature is not too high, which can degrade the product.
Specifically, a carboxyfluorescein succinimidyl ester crude product is dissolved by ethyl acetate, reduced pressure concentration (the vacuum degree is less than or equal to-0.09 MPa) is carried out at 20-50 ℃ until no liquid drops drop, and reduced pressure drying is carried out at 30-50 ℃ for 3-4 h.
The carboxyl fluorescein succinimide ester product prepared by the method has an orange red powder appearance, and the content of DCU is less than 3.0%; the content of NHS and the content of carboxyfluorescein are both less than 0.5 percent; the other single impurity peaks are less than 1.5%, and the total impurity is less than 5.0%. Less than 0.072% of tetrahydrofuran, less than 0.5% of acetone, less than 0.029% of n-hexane and less than 0.5% of methyl tert-butyl ether. The residue on ignition is less than 0.1%.
The invention also protects the application of the solvate in the field of medicines.
The following examples are intended to illustrate the invention in detail, but are not intended to limit the scope of the invention.
Example 1 this example relates to the preparation of a solvate comprising the following steps:
1) condensation: adding 5(6) -carboxyfluorescein (1:1) (15.00g), NHS (5.96g) and THF (75ml) into a 250ml reaction bottle, stirring and dissolving at room temperature, adding DCC (11.51g) in batches at the temperature of 15-20 ℃, and after the addition, keeping the temperature at 20-30 ℃ and stirring for reaction for 2 hours.
2) And (3) column chromatography purification: after the reaction is finished, filtering the reaction solution, rinsing a filter cake with THF (30ml x 2), draining, evaporating THF from the filtrate in a water bath at 30 ℃ under reduced pressure, adding acetone (75ml) into the residue, stirring for 1h at 15-30 ℃, filtering out insoluble substances, adding 100-200 meshes of silica gel (30g) into the filtrate in a water bath at 30 ℃ under reduced pressure, evaporating to dryness, and performing chromatographic separation and purification. Eluent: EA n-hexane 1:1, TLC monitoring the effluent and starting collection when TLC monitoring of the effluent shows a clear target product spot. Collecting the eluent, merging, concentrating under reduced pressure (the vacuum degree is less than or equal to-0.09 MPa) in water bath at 25-30 ℃ until no liquid drops drop, adding methyl tert-butyl ether (150ml) (rho is 0.74) into the residue, pulping for 1h for 20-30 hours until the solid is uniformly dispersed, continuing to distill under reduced pressure in water bath at 25-30 ℃ until the solid is dried, and obtaining orange-red solid.
3) Solvation: dissolving with 150ml ethyl acetate, concentrating under reduced pressure at 25-30 deg.C (vacuum degree is less than or equal to-0.09 MPa) until no liquid drops drop, and drying under reduced pressure at 30-40 deg.C for 3h to obtain 13.25g of orange red powder, i.e. 5(6) -carboxyfluorescein succinimidyl ester ethyl acetate solvate.
The appearance of the obtained solvate is orange red powder, and the content of DCU is 1.0%; NHS content of 0.10%, 5(6) -carboxyfluorescein content of 0.3%; the other single impurity peaks are less than 1.5%, and the total impurity content is 4.0%. Tetrahydrofuran content 0.001%, acetone content 0.01%, n-hexane content 0.001%, and methyl tert-butyl ether content 0.02%. The residue on ignition is less than 0.1%. The content of ethyl acetate was 8.0%, and the mixing ratio of 5-carboxyfluorescein succinimide ester and 6-carboxyfluorescein succinimide ester in 5(6) -carboxyfluorescein succinimide ester was 1: 1.
Example 2 this example relates to the preparation of a solvate comprising the following steps:
1) condensation: adding 5(6) -carboxyfluorescein (1:5) (15.00g), NHS (3.02g) and THF (80ml) into a 250ml reaction bottle, stirring and dissolving at room temperature, adding DCC (9.05g) in batches at the temperature of 30-40 ℃, and after the addition, keeping the temperature at 20-30 ℃ and stirring for reaction for 2 hours.
And (3) post-treatment:
2) and (3) column chromatography purification: after the reaction is finished, filtering the reaction solution, rinsing a filter cake with THF (40ml x 2), draining, evaporating THF in a filtrate water bath at 50 ℃ under reduced pressure, adding acetone (75ml) into the residue, stirring for 1h at 15-20 ℃, filtering out insoluble substances, adding 100-200 meshes of silica gel (30g) into the filtrate, evaporating to dryness at 50 ℃ under reduced pressure, and performing chromatographic separation and purification. Eluent: EA n-hexane 1:3, TLC monitoring the effluent and starting collection when TLC monitoring of the effluent shows a clear target product spot. Collecting the eluent, concentrating under reduced pressure at 30 ℃ (vacuum degree is less than or equal to-0.09 MPa) until no liquid drops drop, adding methyl tert-butyl ether (150ml) into the residue, pulping for 1h until the solid is uniformly dispersed, continuing to carry out water bath at 40-50 ℃, and distilling under reduced pressure until the solid is dried to obtain orange red solid.
3) Solvation: dissolving with 250ml ethyl acetate, concentrating under reduced pressure at 40-50 deg.C (vacuum degree is less than or equal to-0.09 MPa) until no liquid drops drop, and drying under reduced pressure at 40-50 deg.C for 3h to obtain 13.85g of orange red powder, namely 5(6) -carboxyfluorescein succinimidyl ester ethyl acetate solvate.
The appearance of the obtained solvate is orange red powder, and the content of DCU is 1.5%; NHS content of 0.15%, 5(6) -carboxyfluorescein content of 0.2%; the other single impurity peaks are less than 1.5%, and the total impurity content is 3.8%. Tetrahydrofuran content 0.002%, acetone content 0.03%, n-hexane content 0.001%, and methyl tert-butyl ether content 0.03%. The residue on ignition is less than 0.1%. The content of ethyl acetate is 10.2%, and the mixing ratio of 5-carboxyfluorescein succinimide ester and 6-carboxyfluorescein succinimide ester in 5(6) -carboxyfluorescein succinimide ester is 1: 5.
Example 3 this example relates to the preparation of a solvate comprising the following steps:
1) condensation: adding 5(6) -carboxyfluorescein (1:3) (15.00g), NHS (9.00g) and THF (90ml) into a 250ml reaction bottle, stirring and dissolving at room temperature, adding DCC (15.00g) in batches at the temperature of 10-15 ℃, and after the addition, carrying out heat preservation and stirring reaction at 20-30 ℃ for 1 h.
2) And (3) column chromatography purification: after the reaction is finished, filtering the reaction solution, rinsing a filter cake with THF (40 ml. times.2), draining, evaporating THF in a filtrate water bath at 40 ℃ under reduced pressure, adding acetone (75ml) into the residue, stirring for 1h at 30-40 ℃, filtering insoluble substances, adding 100-200 meshes of silica gel (30g) into the filtrate, evaporating to dryness at 40 ℃ under reduced pressure, and performing chromatographic separation and purification. Eluent: EA n-hexane 1:5, TLC monitoring the effluent and starting collection when TLC monitoring of the effluent shows a clear target product spot. Collecting the eluent, concentrating under reduced pressure at 50 ℃ (vacuum degree is less than or equal to-0.09 MPa) until no liquid drops drop, adding methyl tert-butyl ether (150ml) into the residue, pulping for 1h until the solid is uniformly dispersed, continuing to carry out water bath at 30-40 ℃, and distilling under reduced pressure until the solid is dried to obtain orange red solid.
3) Solvation: dissolving with 300ml ethyl acetate, concentrating under reduced pressure at 30-40 deg.C (vacuum degree is less than or equal to-0.09 MPa) until no liquid drops drop, and drying under reduced pressure at 40-50 deg.C for 3h to obtain 14.85g of orange red powder, namely 5(6) -carboxyfluorescein succinimidyl ester ethyl acetate solvate.
The appearance is orange red powder, and the content of DCU is 1.2%; NHS content 0.11%, 5(6) -carboxyfluorescein content 0.1%; the other single impurity peaks are less than 1.5%, and the total impurity content is 3.5%. Tetrahydrofuran content 0.001%, acetone content 0.01%, n-hexane content 0.0009%, methyl tert-butyl ether content 0.04%. The residue on ignition is less than 0.1%. The content of ethyl acetate is 15.2%, and the mixing ratio of 5-carboxyfluorescein succinimide ester and 6-carboxyfluorescein succinimide ester in 5(6) -carboxyfluorescein succinimide ester is 1: 3.
Comparative example 1
The only difference compared to example 1 is that no solvation treatment was performed, i.e. step 3 was omitted), yielding 5(6) -carboxyfluorescein succinimidyl ester.
Comparative example 2
Compared with the example 1, the method is only different in that the condensation reagent DCC is added once in the initial stage of the reaction in the step 1), and the reaction temperature is 40-50 ℃ rather than 20-30 ℃.
Examples of the experiments
The products obtained in examples 1 to 3 and comparative examples 1 to 2 were subjected to dissolution test.
The experimental method comprises the following steps: the products prepared in the examples and comparative examples were placed in a 5ml EP tube, 3.048g of buffer was added, 3200rpm was applied, and the solution was vortexed until it was clear.
The prescription and the preparation method of the buffer solution comprise the following steps: adding sodium dihydrogen phosphate and disodium hydrogen phosphate into a beaker respectively, adding water for injection, and stirring until the sodium dihydrogen phosphate and the disodium hydrogen phosphate are dissolved. The pH value should be 6.8-7.8.
The experimental results are as follows:
| experimental phenomena | Results of the experiment | |
| Example 1 | Gradually dissolve | 2 min dissolving and clearing |
| Example 2 | Gradually dissolve | 2 min dissolving and clearing |
| Example 3 | Gradually dissolve | 2 min dissolving and clearing |
| Comparative example 1 | Largely undissolved | Undissolved clear in 30 minutes |
| Comparative example 2 | Small amount of undissolved | Undissolved clear in 30 minutes |
From the above results, it is understood that the solvate of the present invention has excellent solubility and dispersibility in water, and as described in examples 1 to 3, the solvate can be completely dissolved in water in a short time. In the absence of solvation, dissolution and dispersibility were very poor, and did not clear as observed in comparative example 1 for 30 minutes. In comparative example 2, when the reaction temperature is higher than 20 to 30 ℃ during the reaction, impurities generated due to side reactions affect the dissolution of the substances, and the dissolution and the dispersibility are poor.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (2)
1. A method for preparing a solvate of carboxyfluorescein succinimide ester,
performing condensation reaction by using carboxyl fluorescein and N-hydroxysuccinimide as raw materials to obtain a carboxyl fluorescein succinimide ester primary product; in the process of condensation reaction, N-dicyclohexyl carbodiimide or 1-ethyl-3 (3-dimethylpropylamine) carbodiimide hydrochloride is used as a condensing agent; controlling the temperature of the condensation reaction to be 20-30 ℃;
the method also comprises the operation of separating and purifying the carboxyl fluorescein succinimidyl ester primary product by chromatography after the condensation is finished, and comprises the following steps:
1) purifying the solution obtained after the condensation reaction is finished, mixing the solution with silica gel, and evaporating the mixture to dryness under reduced pressure to obtain silica gel to be treated;
2) eluting the silica gel to be treated by using a mixed solution of ethyl acetate and normal hexane as an eluent, and purifying the eluent to obtain carboxyfluorescein succinimidyl ester; dissolving carboxyfluorescein succinimidyl ester in ethyl acetate, and volatilizing the ethyl acetate; volatilizing the ethyl acetate under the vacuum condition of 20-50 ℃;
the carboxyfluorescein is 5(6) -carboxyfluorescein, and the mass ratio of the 5-carboxyfluorescein to the 6-carboxyfluorescein in the 5(6) -carboxyfluorescein is 1: 1-5;
and/or adding the condensing agent in batches during the reaction;
and/or tetrahydrofuran is used as a reaction solvent in the condensation reaction.
2. The method according to claim 1, wherein the solution obtained after the condensation reaction is purified by the following steps: filtering the obtained solution, evaporating the filtrate at 30-50 ℃ under reduced pressure to remove the solvent, adding acetone into the residue, stirring at 15-40 ℃ for 0.5-1 h, and filtering out insoluble substances;
and/or, the specific steps of purifying the eluent are as follows: concentrating the eluent under reduced pressure at 20-50 ℃ until no liquid drops, adding methyl tert-butyl ether into the residual solid, pulping at 20-40 ℃ until the solid is uniformly dispersed, and distilling under reduced pressure at 20-50 ℃ until the solid is dried.
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