CN101125855B - Method for separating hemporfin isomer and separated isomer - Google Patents
Method for separating hemporfin isomer and separated isomer Download PDFInfo
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- CN101125855B CN101125855B CN2006100301856A CN200610030185A CN101125855B CN 101125855 B CN101125855 B CN 101125855B CN 2006100301856 A CN2006100301856 A CN 2006100301856A CN 200610030185 A CN200610030185 A CN 200610030185A CN 101125855 B CN101125855 B CN 101125855B
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Abstract
The invention belongs to photodynamic treatment field, in particular relates to photosencitizer of photodynamic treatment. The invention provides a reversed-phase purification method for separating a 3 (or 8)-(1-methoxyethyl)-8 (or 3)-(1-ethoxyl) deuteroporphyrin IX positional isomer. The method can be used for preparing an 8-(1- methoxyethyl)-3-(1-ethoxyl) deuteroporphyrin IX or 3-(1-methoxyethyl)-8-(1-ethoxyl) deuteroporphyrin IX single isomer. The two isomers, through an animal experiment, are proved to be applicable to blocking new-born veins effectively as the photosencitizer. The single isomer of the invention can be used for preparing medicine with single molecule structure and steadier properties.
Description
Technical field
The present invention relates to field of photodynamic, more specifically, the present invention relates to a kind of photosensitizers of optical dynamic therapy.
Background technology
Optical dynamic therapy (PDT) imposes Photoactive compounds to the patient; Photosensitizers is in the enrichment of new vessel position; With low-intensity laser irradiation, excite photosensitizers to make it to produce photochemical reaction again, photochemically reactive position generation radical is taking place; Can kill and wound the histocyte at this position, mainly produce result of treatment through direct CDCC and sealing lesions position blood vessel thereof.
The photosensitizers that is used for optical dynamic therapy has hematoporphyrin derivative (HPD), benzoporphyrin derivative (BPD), chlorin e 6 monoammonium aspartate acid amides (Npe6), sulfonic acid aluminium phthalocyanine (CASPc), intermediary four (hydroxy phenyl)-chlorin (mTHPC), tin ethyl just C.I. Natural Red 8 (SnEt2), 5-amino-laevulic acid (ALA) etc., is used for tumour, dermopathic optical dynamic therapy.
3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX (hemporfin) is a kind of effective new type light dynamic therapy medicine; Some cancer and nevus flammeus there are good curing effect (Xu Deyu, Chen Wenhui, Shen Nianci: light power cancer curing medicine 3-or 8-3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX; The Chinese laser medical journal; 1993,2:1; Gu Ying, Liu Fanguang, Wang Kai etc., blood quinoline methyl ether are used for the clinical study of PDT treatment nevus flammeus, Chinese laser medical journal 2000,9 (3): 185).
The photosensitizers that is used for optical dynamic therapy at present, major part are complicated mixtures, form uncertainly, structure is still disputable, have influenced the stability of medication preparation, result of treatment.Optical dynamic therapy medicine 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) the NSC 19663 IX (hemporfin) that uses at present is as single compound; Has clear and definite chemical structure; But still comprise 2 kinds of positional isomerss; Its shortcoming that has is that character is stable inadequately, thereby has influence on the safety of effect.The medicine single in order to obtain molecular structure, that character is more stable is necessary further to separate 2 kinds of positional isomerss, is used for more effectively the patient being carried out optical dynamic therapy.
Fractionation to the hemporfin positional isomers remains difficult point of the prior art, need take all factors into consideration a large amount of factors such as method for splitting, splitting condition (comprising pH value, temperature, time etc.), resolution reagent that should take.So far also do not obtain such isolating, that molecular structure is single and character is more stable medicine in the prior art at present.
Summary of the invention
One of the object of the invention provides a kind of method of separation 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX (hemporfin) positional isomers.
Another object of the present invention provides a kind of single compound 8-of molecular structure (1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX that obtains according to said method (hemporfin isomer-A).
Another object of the present invention provides a kind of single compound 3-of molecular structure (1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX that obtains according to said method (hemporfin isomer-B).
Two kinds of isomer adopt the HPLC method to separate:
3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX position different structure mixture uploads to the reverse phase silica gel post,, detects at the 395nm place as the moving phase wash-out with the unit alcohol of C1～C3 or THF, collects eluting peak.
Said reverse phase silica gel can be selected a kind of in C18 silica gel, C8 silica gel, C4 silica gel or the phenyl silica gel for use, preferred C8 silica gel; The particle diameter of reverse phase silica gel is 10～150 μ m, preferable particle size 30～70 μ m; Aperture 60～the 200A of reverse phase silica gel, preferred aperture 60A.
The moving phase particular methanol, preferred, moving phase is the methanol solution that contains damping fluid, pH4～5.Wherein buffered soln is selected from acetic acid-sodium-acetate, acetic acid-ammonium acetate, phosphoric acid-phosphoric acid salt etc., preferred acetic acid-ammonium acetate buffer.
3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX is mixed with the sample solution of 5mg/mL concentration with the dissolving of HPLC moving phase, filters the back sample introduction.
MOS-Hypersil (C
8), 10 * 250mm, with methyl alcohol-1.5M acetate--amine acetate damping fluid (pH4.76) is a moving phase (65:35V/V), and at flow velocity 2mL/min, post is pressed under the 125Bar condition so adopt reversed-phased high performace liquid chromatographic to separate.
Use the DAD detector,, collect eluting peak detecting wavelength: 395nm detection.Have 2 eluting peaks, isomer A is the short component of HPLC RT, and isomer B is the long component of HPLC RT.
Isomer A flow point, the isomer B flow point collected are used with quadrat method and are handled.Can adopt organic solvent extractionprocess and direct method of enrichment, preferred organic solvent extractionprocess, extraction solvent is selected from methyl acetate, ETHYLE ACETATE, sherwood oil or ether etc.; Organic solvent extraction liquid removes solvent under reduced pressure to doing, and promptly gets the sample of wanting.
Specifically can adopt following method: collect sample and pour in ETHYLE ACETATE (or ether)/zero(ppm) water two-phase solution; Organic phase is obtained in jolting, with the distilled water wash organic phase for several times; Organic phase removes solvent under reduced pressure to doing under 40 ℃; Put in the vacuum drier,, obtain single isomer sample with the Vanadium Pentoxide in FLAKES drying under vacuum overnight.
8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX has the structure shown in the formula (I):
Hemporfin isomer A
3-(1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX has the structure shown in the formula (II):
Hemporfin isomer B
Description of drawings
Fig. 1 is the HPLC collection of illustrative plates of 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX.
Fig. 2 A is 3-behind the purifying (1-methoxy ethyl)-pure article HPLC collection of illustrative plates of 8-(1-hydroxyethyl) NSC 19663 IX (hemporfin isomer B).
Fig. 2 B is 8-behind the purifying (1-methoxy ethyl)-pure article HPLC collection of illustrative plates of 3-(1-hydroxyethyl) NSC 19663 IX (hemporfin isomer A).
Fig. 3 A is HPLC collection of illustrative plates and two kinds of content of isomer of 5 minutes hepatic tissue samples after the rat administration.
Fig. 3 B is HPLC collection of illustrative plates and two kinds of content of isomer of 30 minutes hepatic tissue samples after the rat administration.
Fig. 3 C is HPLC collection of illustrative plates and two kinds of content of isomer of 8 hours hepatic tissue samples after the rat administration.
Embodiment
The inventor isolates 2 kinds of positional isomerss first through a large amount of work from 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX, it is single to have obtained molecular structure, the medicine that character is more stable.
And the inventor finds that the isomer A metabolism of hemporfin is very fast, and liver is accumulated few, uses separately to have lower liver toxicity than mixtinite, can reduce such untoward reaction; And the isomer B of hemporfin is higher in liver concentration, and the residence time is long, and more helping with the liver is the optical dynamic therapy effect of target, like the optical dynamic therapy of liver cancer.
Photoactive compounds
The chemical structure conclusive evidence of two kinds of isomer adopts one dimension NOE difference spectrum (1D-NOEDIFF) technology, and the result is following:
Isomer A: the chemical shift of hydrogen spectrum is broadly divided into three parts: δ 10-11ppm is fragrant proton signal; δ 2-7ppm is the aliphatics proton signal; δ-3.98ppm is NH proton signal in the ring, and this is owing to the influence that just shielded by fragrant circulation appears at unusual High-Field.On the basis of aforementioned hemporfin nuclear magnetic resonance spectroscopy test report, isomer A is carried out spectral line ownership: δ 10.78 (H-5); δ 10.54 (H-10); δ 10.28 (H-15); δ 10.23 (H-20); δ 6.54 (
CHOH); δ 6.15 (
CH-OCH
3); δ 4.35 (C
25H
2, C
27H
2); δ 3.73 (C
2-CH
3); δ 3.69 (C
7-CH
3); δ 3.64 (C
12-CH
3); δ 3.62 (C
18-CH
3); δ 3.56 (OCH
3); δ 3.22 (C
26H
2, C
28H
2); δ 2.16 (CH
CH 3 ); δ-3,98 (NH).
The experimental result of isomer A one dimension NOE difference spectrum: irradiation δ 10.78, then δ 6.54, δ 3.69, δ 2.16 produce the NOE gain; Irradiation δ 10.54, then δ 6.15, δ 3.64, δ 3.56, δ 2.16 produce the NOE gain; Irradiation δ 10.28, then δ 4.35, δ 3.22 produce the NOE gain; Irradiation δ 10.23, then δ 3.73, δ 3.62 produce the NOE gain. these digital proof isomer A substituting group CHOHCH
3Be connected on the C-8 position CH-(OCH
3) CH
3Be connected on the C-3 position.Irradiation 66.65, δ 6.15, δ 4.35, δ 3.56, δ 3.22 and δ 2.16 also obtain experimental result as above respectively.
Can confirm that said isomer A is 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX, for molecular structure is a following formula:
Hemporfin isomer A
Isomer B: the chemical shift of hydrogen spectrum is broadly divided into three parts: δ 10-11ppm is fragrant proton signal; δ 2-7ppm is the aliphatics proton signal; δ-3.98ppm is NH proton signal in the ring, and this is owing to the influence that just shielded by fragrant circulation appears at unusual High-Field.On the basis of aforementioned hemporfin nuclear magnetic resonance spectroscopy test report, the spectral line of isomer B ownership: δ 10.70 (H-5); δ 10.57 (H-10); δ 10.28 (H-15); δ 10.25 (H-20); δ 6.13 (
CHOH);
δ6.55(
CH-OCH
3)；δ4.35(C
25H、C
27H
2)；δ3.58—3.75(C
7-CH
3、C
2-CH
3、C
18-CH
3)；δ3.56、3.53(C
12-CH
3)；δ3.38(OCH
3)；δ3.21(C
26H
2、C
28H
2)；δ2.16(CH
CH 3)；δ-3.95(NH)。
The experimental result of isomer B one dimension difference spectrum: irradiation δ 10.70, then δ 6.55, δ 3.64, δ 3.62, δ 3.38 produce the NOE gain; Irradiation δ 10.57, then δ 6.13, δ 3.71, δ 3.68 produce the NOE gain; Irradiation δ 10.28 and δ 10.25, then δ 4.35, δ 3.71, δ 3.68, δ 3.64, δ 3.62 produce the NOE gain; Irradiation δ 6.55, then δ 10.7, δ 3.71, δ 3.68, δ 3.38, δ 2.16 produce the NOE gain; Irradiation δ 6.13, then δ 10.57, δ 3.71, δ 3.68, δ 3.56, δ 3.53 produce the NOE gain.Irradiation δ 4.35, δ 3.69, δ 3.60, δ 3.53, δ 3.21 and δ 2.16 also obtain experimental result as above respectively.
Can confirm that said isomer B is 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX, molecular structure is a following formula:
Hemporfin isomer B
Treatment mechanism
The present invention relates to form the disease that causes with optical dynamic therapy because of deleterious new vessel, the photodynamic therapy scheme makes deleterious neovascularization reduce.
In the method for the invention, giving needs the patient of treatment to take suitable Photoactive compounds, presents in an amount at least sufficient to make the Photoactive compounds of patient's intralesional to reach effective concentration.Take after after a while, make the compound of effective concentration accumulate in focus after, use the rayed that is absorbed by this Photoactive compounds should the zone.Photoactive compounds is produced active oxygen and radical by optical excitation, causes lesion region cell photochemical damage, the new vessel of sealing diseased region, thereby the various diseases that treatment causes because of harmful vascularization.
Administration and dosage
Photoactive compounds can various administrations, like oral, parenterai administration or rectal administration, are advisable with parenterai administration, like intravenously, intramuscular or subcutaneous injection.With intravenous injection is best.
The dosage of Photoactive compounds can be according to administering mode, preparation type, whether coupling target part has very big variation.It is generally acknowledged, relevant between preparation, administering mode and the dosage level of optical active matter.Usually; The typical doses scope of 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX or 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX is 0.1-100mg/kg; Preferable dosage range is 0.5-50mg/kg, and better dosage range is 1-10mg/kg.Those skilled in the art also can confirm proper dosage through experiment.
Being used for various parameters effective, the selective light photodynamic therapy in the present invention is to be mutually related.Therefore, can be with respect to other parameters (for example in the photodynamic therapy timed interval between employed optical throughput, illuminance, time length and dosage, administration and the rayed etc.) and adjustment dosage.The obvious damage of using these parameters all should adjust to significantly improve eyesight and not producing the normal eyes tissue is advisable.Those skilled in the art can confirm suitable parameters through experiment.
In other words, when the dosage of Photoactive compounds reduced, the optical throughput of sealing choroidal neovascular tissue had the trend of increase; Otherwise need to reduce optical throughput, then need increase the dosage of Photoactive compounds or increase the enrichment degree that the promotion of target property increases the diseased region Photoactive compounds.
The light treat-ment
Some term of light treatment relating to parameters is different in different authors and publication.Such as optical throughput, also claim light dosage, optical energy density; Illuminance is also claimed power level, power density.These terms are that those skilled in the art uses and understands, and explain at this.
After the Photoactive compounds administration, the target tissue of eye is shone under selecteed drug absorption wavelength.For hematoporphyrin monomethyl ether, selected wavelength region about 630 ± 20nm, more preferably is that the penetration power of this range of wavelength in body tissue is relatively good about 630 ± 10nm generally.
The result of irradiation causes Photoactive compounds to be in excited state, and interacts with other compounds, forms singlet oxygen (Singlet Oxygen) and other radicals, causes the structure deteriorate of blood vessel epithelial cell.Singlet oxygen and other radical main damage membrane structures comprise cytolemma, mitochondrial membrane, lysosome membrane and nuclear membrane.The damage of blood vessel epithelial cell causes follow-up platelet aggregation, takes off particle and thrombosis, causes the obstruction and the sealing of blood vessel.
According to the type, the target tissue degree of depth of tissue and the amount of fluid or blood on it, the optical throughput of irradiation can have very cataclysm.But that preferable is 50-200 joule/cm
2
Illuminance generally changes in 50-800mW/cm
2, with about 100-600mW/cm
2For good.Yet, select to use higher illuminance, can shorten treatment time and reach same effect.
After the Photoactive compounds administration to the interbody spacer the most in good time between the light treatment also according to administering mode, form of medication and preparation type and different.The timed interval after the photosensitizers administration, preferable was 5-30 minute from 1 minute to 2 hours, and better is 10-25 minute.
Medicable disease
3-provided by the invention (1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX or 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX; Be used to prepare treatment and form the medicine of the disease that causes because of deleterious new vessel, this medicine forms the photosensitizers of the disease that causes because of deleterious new vessel as optical dynamic therapy.
Because of forming the disease that causes, deleterious new vessel comprises: dermatosis, like nevus flammeus; Also comprise ophthalmic diseases, like degeneration of macula.
The evaluation of treatment
The damage of the effect of optical dynamic therapy available histology sections observation endotheliocyte on animal model and the encapsulation situations of lesions position new vessel.Typically, the destruction of new vessel shows as formation of vacuoles in the vascular endothelial cell endochylema, and the nucleus shrinkage is unusual, the formation of visible platelet aggregation and blood clot in the lumen of vessels.
The blood vessel sealing that is positioned at skin can the direct viewing effect; Be positioned at the blood vessel sealing of organizing deep layer, eye choroid for example, available angiography technology is observed neovascularity and is reduced.
The distribution of different isomerization body
Liver is the main distribution organ of hemporfin, and most of medicine excretes from bile through liver.Simultaneously, porphyrin class medicine main adverse reactions is possibly cause dysfunction of liver, can show as transaminase and raise.The inventor finds that the isomer A metabolism of hemporfin is very fast, and liver is accumulated few, uses separately to have lower liver toxicity than mixtinite, can reduce such untoward reaction.
Simultaneously, the isomer B of hemporfin is higher in liver concentration, and the residence time is long, and it is the optical dynamic therapy effect of target that prompting more helps with the liver, like the optical dynamic therapy of liver cancer.
Therefore, when practical implementation is treated, can select to use the isomer A or the isomer B of hemporfin, to reach best result of treatment according to patient's situation.
Major advantage of the present invention is:
(1) a kind of method of separating 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) NSC 19663 IX (hemporfin) positional isomers is provided first, it is single that this method can obtain molecular structure easily, the hemporfin medicine that character is more stable.
(2) find that first the isomer A metabolism of hemporfin is very fast, liver is accumulated few, uses separately to have lower liver toxicity than mixtinite; And the isomer B of hemporfin is higher in liver concentration, and the residence time is long, and more helping with the liver is the optical dynamic therapy effect of target.Thereby when adopting hemporfin to treat, can select to use the isomer A or the isomer B of hemporfin according to patient's situation.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.
Embodiment 1: the preparation of hemporfin
1.3 (8)-(1-methoxy ethyl) preparation of-8 (3)-(1-hydroxyethyl) NSC 19663 IX bullions
In the acidproof lass lining reactor of 50L, adding volume ratio is the methanol mixed solution 30L of 3:1; Stir down and slowly splash into by Liquid titrator; By the method for Chinese patent 01105208.2 specification sheets embodiment 1 record prepare 3; 8-two-(1-bromotrifluoromethane)-NSC 19663 IX hydrobromate Glacial acetic acid min. 99.5 saturated solution 10L, the control rate of addition makes reaction mixture temperature remain on 10～20 ℃.Dropwise, reaction solution continues to stir 2h, places 4h.The dropping 10N NaOH aqueous solution to reaction solution is strong basicity (about PH13) in stirring down then, places more than the 10h, adds acetic acid to be neutralized to PH4～5; Add the water dilution of 5 times of volumes, placement is spent the night, the throw out natural sedimentation; Incline after the supernatant suction strainer collecting precipitation, water thorough washing; Drain, put and use P in the vacuum drier
2O
5Vacuum-drying.Get auburn 3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) the NSC 19663 IX (53.3%HPLC), 3 that contain; 8-two-(1-methoxy ethyl)-NSC 19663 IX (19.5%HPLC) and 3; Bullion 160 grams of 8-two-(1-hydroxyethyl)-NSC 19663 IX (27.1%HPLC), yield 80%.
2.3 the chromatographic separation and purification of-8 (3)-(1-hydroxyethyl) NSC 19663 IX (8)-(1-methoxy ethyl)
Get 10 of 60 * 600mm glass chromatography columns, add the activated silica gel of 200g respectively, carefully strike reality.Be added on the silicagel column upper strata after 3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) NSC 19663 IX bullion 6g and the 30g silica gel H of getting a step preparation grinds well, and add silica gel H 20g in addition above that, ditto strike reality.Add developping agent (methyl alcohol-chloroform-formic acid (10:1:0.1)) and carry out chromatographic separation, the Fractional Collections flow point detects with thin-layer chromatography earlier; When the TLC detection shows that required component is single spot, promptly carry out HPLC and detect, merge the flow point that HPLC analyzes relative peak area >=95% of RT 3.43min; Washing; The organic phase evaporated under reduced pressure promptly gets, 3 (8)-(1-methoxy ethyl) the about 10g of-8 (3)-pure article of (1-hydroxyethyl) NSC 19663 IX (hemporfin) (yield 16.7%).
Embodiment 2: the HPLC of hemporfin positional isomers separates
Instrument: Agilent1100 type high performance liquid chromatograph; The DAD detector detects wavelength: 395nm
Moving phase: methyl alcohol-1.5M acetate--amine acetate damping fluid (pH4.76) (65:35V/V)
Chromatographic column: MOS-Hypersil (C
8), 10 * 250mm
Flow velocity: 2mL/min
Post is pressed: 125Bar
Get 100 milligrams in the hemporfin sample of the foregoing description 1 preparation,, be mixed with the sample solution of 5mg/mL concentration, filter before the sample introduction with the dissolving of HPLC moving phase.Each above-mentioned solution 400 microlitres of sample introduction behind the sample introduction, are collected flow point A respectively according to HPLC color atlas (see figure 1), RT be 11.94 minutes be 14.35 minutes with flow point B RT.Accumulative total 40 times is collected flow point A136ml, flow point B133ml altogether.Collect liquid and pour ETHYLE ACETATE (or ether) into--in the zero(ppm) water two-phase solution, organic phase is obtained in jolting, and with distilled water wash organic phase 3 times, organic phase removes solvent under reduced pressure to doing under 40 ℃, put in the vacuum drier, with the Vanadium Pentoxide in FLAKES drying under vacuum overnight.
As a result, get hemporfin positional isomers A36.2 milligram, isomer B35.0 milligram respectively.
The HPLC collection of illustrative plates of 3-behind the purifying (1-methoxy ethyl)-pure article of 8-(1-hydroxyethyl) NSC 19663 IX (hemporfin isomer B) is seen Fig. 2 A.
HPLC collection of illustrative plates Fig. 2 B of 8-behind the purifying (1-methoxy ethyl)-pure article of 3-(1-hydroxyethyl) NSC 19663 IX (hemporfin isomer A).
Embodiment 3: the preparation scale of hemporfin positional isomers separates
Separation system adopts Flash150M, built-in prepackage C
8Chromatography column (150mm ID * 30cm), produce by Biotage company (U.S.), the silica gel particle diameter is 35～70 μ m, the aperture is 60A.
Sample is prepared: get 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-pure article of (1-hydroxyethyl) NSC 19663 IX (content>95%), 30 grams of embodiment 1 preparation, use the THF dissolution filter, add C8 reverse phase silica gel 100 and restrain; Concentrating under reduced pressure; Evaporate to dryness among the appearance post SIM, is prepared against last appearance in the adding.
Last appearance and collection: with methyl alcohol-1.5M acetate--amine acetate damping fluid (pH4.76) is a moving phase (65:35V/V); Above-mentioned sample is passed through last appearance post SIM; Bring in the chromatography column and separate; Flow rate control is at 200ml/min, and detector wavelength 395nm collects hemporfin isomer-A flow point [8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX] and hemporfin isomer-B flow point [3-(1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX].
Aftertreatment: flow point is collected liquid and is used ethyl acetate extraction, and again with organic phase concentrating under reduced pressure, evaporate to dryness, drying under reduced pressure promptly gets sample, hemporfin positional isomers A11.8 gram, isomer B11.3 gram.
Embodiment 4: the structural identification (nuclear magnetic resonance spectrum) of hemporfin isomer A and isomer B
1. instrument and method
Instrument: Varian UNITY Inova600 NMR
Solvent: DMSO-d6
Interior mark: TMS
2. resolve
Hemporfin isomer A and isomer B see from chemical structure, only is due to last two substituting groups in C-3 and the C-8 position difference of exchanging.This research is adopted one dimension NOE difference spectrum technology (1D-NOEDIFF) that these two isomer are carried out structure and is identified.
2.1. the chemical shift of hydrogen spectrum is broadly divided into three parts: δ 10-11ppm is fragrant proton signal; δ 2-7ppm is the aliphatics proton signal; δ-3.98ppm is NH proton signal in the ring, and this is owing to the influence that just shielded by fragrant circulation appears at unusual High-Field.
2.2. the structure of isomer A demonstration
On the basis of hemporfin nuclear magnetic resonance spectroscopy test report, isomer A is carried out spectral line ownership: δ 10.78 (H-5); δ 10.54 (H-10); δ 10.28 (H-15); δ 10.23 (H-20); δ 6.54 (
CHOH); δ 6.15 (
CH-OCH
3); δ 4.35 (C
25H
2, C
27H
2); δ 3.73 (C
2-CH
3); δ 3.69 (C
7-CH
3); δ 3.64 (C
12-CH
3); δ 3.62 (C
18-CH
3); δ 3.56 (OCH
3); δ 3.22 (C
26H
2, C
28H
2); δ 2.16 (CH
CH 3 ); δ-3,98 (NH).
2.3. the one dimension NOE of isomer A difference spectrum
Irradiation δ 10.78, then δ 6.54, δ 3.69, δ 2.16 produce the NOE gain; Irradiation δ 10.54, then δ 6.15, δ 3.64, δ 3.56, δ 2.16 produce the NOE gain; Irradiation δ 10.28, then δ 4.35, δ 3.22 produce the NOE gain; Irradiation δ 10.23, then δ 3.73, δ 3.62 produce the NOE gain.These digital proof isomer A substituting group CH-(OCH
3) CH
3Be connected on the C-8 position CHOHCH
3Be connected on the C-3 position.Irradiation δ 6.65, δ 6.15, δ 4.35, δ 3.56, δ 3.22 and δ 2.16 also obtain experimental result as above respectively.
2.4. the spectral line of isomer B ownership
δ10.70(H-5)；δ10.57(H-10)；δ10.28(H-15)；δ10.25(H-20)；δ6.13(
CHOH)；δ6.55(
CH-OCH
3)；δ4.35(C
25H、C
27H
2)；δ3.58-3.75(C
7-CH
3、C
2-CH
3、C
18-CH
3)；δ3.56、3.53(C
12-CH
3)；δ3.38(OCH
3)；δ3.21(C
26H
2、C
28H
2)；δ2.16(CH
CH 3)；δ-3.95(NH)。
2.5. isomer B one dimension NOE difference spectrum
Irradiation δ 10.70, then δ 6.55, δ 3.64, δ 3.62, δ 3.38 produce the NOE gain; Irradiation δ 10.57, then δ 6.13, δ 3.71, δ 3.68 produce the NOE gain; Irradiation δ 10.28 and δ 10.25, then δ 4.35, δ 3.71, δ 3.68, δ 3.64, δ 3.62 produce the NOE gain; Irradiation δ 6.55, then δ 10.7, δ 3.71, δ 3.68, δ 3.38, δ 2.16 produce the NOE gain; Irradiation δ 6.13, then δ 10.57, δ 3.71, δ 3.68, δ 3.56, δ 3.53 produce the NOE gain.These NOE digital proof isomer B substituting group CHOHCH
3Be connected on the C-8 position CH-(OCH
3) CH
3Be connected on the C-3 position.Irradiation δ 4.35, δ 3.69, δ 3.60, δ 3.53, δ 3.21 and δ 2.16 also obtain experimental result as above respectively.
3. conclusion
3.1. the chemical structural formula of hemporfin isomer A is accredited as following formula, that is, and and 3-(1-hydroxyethyl)-8-(1-methoxy ethyl)-NSC 19663 IX.
3.2. the chemical structural formula of hemporfin isomer B is accredited as following formula, that is, and and 3-(1-methoxy ethyl)-8-(1-hydroxyethyl)-NSC 19663 IX.
Embodiment 5: hemporfin and positional isomers thereof are to the light power fragmentation test of vascular endothelial cell
Get the good mouse pulmonary vascular endothelial cell (RPMVEC of growth conditions; Be called for short EC, prepare by following literature method: Chen SF, Fei X; Li SH.A new simple method for isolation ofmicrovascular endothelial cells avoiding both chemical and mechanicalinjuries.Microvas Res; 1995,50:119), after 0.3% trysinization; Blow and beat into single cell suspension, cell density is adjusted into 5.0 * 10 with the DMEM nutrient solution that contains 5% serum
4Individual/ml, and it is inoculated on the 96 porocyte culture plates, every hole adds 200 μ l, place incubator to hatch 18h after, the nutrient solution in each hole of sucking-off.Hemporfin, isomer A, isomer B with the foregoing description 1, embodiment 2 preparations; Three kinds of photosensitizerss are mixed with the solution that concentration is respectively 0.75 μ g/ml, 0.625 μ g/ml, 0.5 μ g/ml, 0.375 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.05 μ g/ml with the DMEM nutrient solution that does not contain serum; Under the lucifuge condition; Every hole adds 200 μ l, each concentration point 6 hole, and incubation time is 4h.Utilize copper vapor laser to shine, power density is 20mw/cm
2, the time is 1000 seconds, energy density is 20J/cm
2It is placed on and hatches 24h in the incubator, in every hole, adds the MTT (being mixed with 5mg/ml with PBS solution) of 20 μ l again, after incubator is hatched 4h, and careful sucking-off nutrient solution and MTT; Every hole adds the DMSO of 150 μ l, place incubator to hatch 12h after, before ELIASA is measured, vibrated 5 minutes; Set the parameter of ELIASA, select 595nm as measuring wavelength, 655nm is wavelength as a reference; Measure optical density(OD) (OD) value in each hole, print result is asked its MV.The experiment triplicate.
After recording the OD value in each hole, ask its cell survival rate, formula is following:
Cell survival rate (%)=(experimental group OD-background group OD)/(blank control group OD-background group OD fully) * 100%
Statistical procedures adopts Stata6.0 software (U.S. Computer Resource Center development), gets the MV of each concentration point cell survival rate of three experiments and does the curve match, thereby calculate its IC
50
These three kinds of photosensitizerss of experiment proof all do not have dark toxicity to EC.Under the copper vapor laser irradiation, hemporfin isomer-A, hemporfin isomer-B and hemporfin are to the medium lethal dose(LD&-{50}) (IC of EC
50) be respectively 0.368 μ g/ml, 0.412 μ g/ml, 0.493 μ g/ml.Two kinds of isomer of hemporfin are similar with the photodynamic killing effect intensity to endotheliocyte that hemporfin is mediated.
6: two kinds of isomer of embodiment are in the distribution of liver
Get 18 of rats, body weight 210.0 ± 4.8g is divided into 3 groups at random, and 6 every group, male and female half and half.Fasting but after can freely drinking water 10 hours, intravenous injection is the 10mg/kg hemporfin (iv) is respectively at after the administration 5,30 and 480min, by femoral artery sacrificed by exsanguination animal.Get blood plasma 0.5ml, and obtain hepatic tissue immediately.Take by weighing the about 0.3g of tissue (precision is weighed), after adding the 1.0ml tri-distilled water and processing homogenate, with 3ml acetonitrile precipitation albumen (after wherein liver tissue homogenate extracts; Sample introduction after dilution), in the centrifugal 5min of 2500rpm, get supernatant 0.5ml; In the centrifugal 10min of 15000rpm; Behind twice high speed centrifugation, get 160 μ l supernatants in the automatic sampling bottle, 20 μ l sample introductions.Measure two kinds of isomer concentration in the tissue with the HPLC method.
After the rat administration 5,30 and 480min after the HPLC collection of illustrative plates of hepatic tissue sample respectively shown in Fig. 3 A, Fig. 3 B and Fig. 3 C.By figure can find out, after the administration in the hepatic tissue content of isomer B be higher than isomer A, administration only detects isomer B in the hepatic tissue after 8 hours.Therefore two isomer are in the difference that is distributed with of liver, and isomer B is higher in the liver abundance, thereby better for the treatment liver cancer efficacy.
Embodiment 7: hemporfin and positional isomers thereof are to the light power damage test of cockscomb skin
Choose totally 30 of body weight 0.8～1.2kg, the prosperous chickens of male Lay in 12～16 weeks of age in week, be divided into 3 groups at random.Every treated animal at first intramuscular injection fiber crops protects that quiet (0.10～0.12mg/kg) anesthesia is that hemporfin, isomer A, the isomer B photosensitizers of 10mg/ml injected into the prosperous chicken vein of Lay by the dosage of 20mg/kg with concentration under the lucifuge condition.Fixedly behind the cockscomb, the mark illumination area, every cockscomb is set 4 irradiated regions, and each irradiated region diameter is 1cm.Select copper vapor laser output mixed light vertically to be shone by optical fiber output, power density is 100mw/cm
2, irradiation time is 20 minutes, the irradiation place does not suitably hide.All use resistance dynamometer to detect output rating before and after the irradiation, guarantee that its fluctuation range is between ± 5%.Control group and each experimental group all behind irradiation, go ahead of the rest when 3 days and 14 days color and the surface condition of visual inspection, recording processing position cockscomb; Cutting organizing of cockscomb illumination area then places 10% formalin fixing immediately; Its pathological change is observed, is write down in conventional dehydration, specimens paraffin embedding slices, HE dyeing under the light microscopic.
According to the criteria for classifying of cockscomb skin morphological change behind the photodynamic action, with cockscomb skin target tissue and non-target tissue to OPK reaction be divided into gently (L, light reaction), in (M, middlereaction), heavy (S, sever reaction) three degree.Adopt X
2Between organizing, check relatively, sets P<0.05 be significant difference.Statistical procedures adopts Stata software.
Three kinds of photosensitizers comparative experiments results that photosensitive damage changes substantially to cockscomb skin target tissue and non-target tissue see the following form:
Photosensitive damage changes characteristics substantially to cockscomb skin relatively sees table 1 for three kinds of photosensitizerss.
Table 1
The result that the light microscopic of the histopathologic slide of three groups of photosensitizerss is observed down shows: the papillary layer capillary network reduces; Tube chamber is little; It is thus clear that new capillary vessel, epidermal area and corium deep layer do not have change, reticular layer vasodilation; Can both produce effective damage effect to target tissue, and all have comparatively clear and definite selectively acting.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (4)
1. a method of separating 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX and 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX positional isomers is characterized in that, comprises the steps:
(1) 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) NSC 19663 IX and 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) NSC 19663 IX mixture are uploaded to the reverse phase silica gel post;
(2) with the methanol solution that contains damping fluid, pH4～5 are as the moving phase wash-out;
(3) detect at the 395nm place, collect eluting peak;
Wherein, described reverse phase silica gel is selected from: C8 silica gel.
2. the method for claim 1 is characterized in that, the particle diameter of reverse phase silica gel is 10～150 μ m, and the aperture is 60～200.
3. method as claimed in claim 2 is characterized in that, the reverse phase silica gel particle diameter is 30～70 μ m, and the aperture is 60.
4. the method for claim 1 is characterized in that, described damping fluid is 1.5M acetate a--ammonium acetate buffer, and the volume ratio of methyl alcohol and acetate--ammonium acetate buffer is 65: 35, and pH 4.76.
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