CN102603753B - Isomers of hemoporfin - Google Patents
Isomers of hemoporfin Download PDFInfo
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- CN102603753B CN102603753B CN201210032807.4A CN201210032807A CN102603753B CN 102603753 B CN102603753 B CN 102603753B CN 201210032807 A CN201210032807 A CN 201210032807A CN 102603753 B CN102603753 B CN 102603753B
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- China
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- isomer
- deuteroporphyrin
- hydroxyethyl
- hemporfin
- methoxy ethyl
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- KFKRXESVMDBTNQ-UHFFFAOYSA-N 3-[18-(2-carboxylatoethyl)-8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-22,23-dihydroporphyrin-21,24-diium-2-yl]propanoate Chemical class N1C2=C(C)C(C(C)O)=C1C=C(N1)C(C)=C(C(O)C)C1=CC(C(C)=C1CCC(O)=O)=NC1=CC(C(CCC(O)=O)=C1C)=NC1=C2 KFKRXESVMDBTNQ-UHFFFAOYSA-N 0.000 title abstract 2
- 239000003814 drug Substances 0.000 claims abstract description 16
- PJUNPWGAFNXSHV-WORMITQPSA-N COC(C)C=1C(=C2NC1C=C1C=C(C(=N1)C=C1C=CC(N1)=CC=1C=CC(N1)=C2)C(C)O)[2H] Chemical compound COC(C)C=1C(=C2NC1C=C1C=C(C(=N1)C=C1C=CC(N1)=CC=1C=CC(N1)=C2)C(C)O)[2H] PJUNPWGAFNXSHV-WORMITQPSA-N 0.000 claims abstract description 14
- 238000002560 therapeutic procedure Methods 0.000 claims description 18
- VAJVGAQAYOAJQI-UHFFFAOYSA-N 3-[18-(2-carboxylatoethyl)-3,8,13,17-tetramethyl-22,23-dihydroporphyrin-21,24-diium-2-yl]propanoate Chemical compound N1C(C=C2C(C)=CC(N2)=CC=2C(=C(CCC(O)=O)C(=C3)N=2)C)=CC(C)=C1C=C1C(C)=C(CCC(O)=O)C3=N1 VAJVGAQAYOAJQI-UHFFFAOYSA-N 0.000 claims description 17
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- MHIITNFQDPFSES-UHFFFAOYSA-N 25,26,27,28-tetrazahexacyclo[16.6.1.13,6.18,11.113,16.019,24]octacosa-1(25),2,4,6,8(27),9,11,13,15,17,19,21,23-tridecaene Chemical class N1C(C=C2C3=CC=CC=C3C(C=C3NC(=C4)C=C3)=N2)=CC=C1C=C1C=CC4=N1 MHIITNFQDPFSES-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the field of photodynamic therapy and particularly relates to photosensitizers for the photodynamic therapy. The invention provides two IX-site single isomers of hemoporfin, namely, 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX and 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX, wherein the isomers are obtained by adopting a reversed-phase purification and separation method. Proved by animal experiments, the two isomers are equally effective in sealing off new vessels when serving as the photosensitizers. The single isomers provided by the invention can be used for preparing a drug with single molecular structure and more stable property.
Description
The present invention is that application number is the divisional application of the application for a patent for invention of CN200610030185.6.
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 patient, photosensitizers is in the enrichment of new vessel position, again with low-intensity laser irradiation, excite photosensitizers to make it to produce photochemical reaction, there is photochemically reactive position generation free radical, can kill and wound the histocyte at this position, mainly produce result for the treatment of by direct cytotoxicity and sealing lesions position blood vessel thereof.
There are just C.I. Natural Red 8 (SnEt2), 5-ALA (ALA) etc. of 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 for the photosensitizers of optical dynamic therapy, for tumour, dermopathic optical dynamic therapy.
3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) deuteroporphyrin IX is a kind of effective novel optical dynamic therapy medicine, some cancer and nevus flammeus are had to good therapeutic action (Xu Deyu, Chen Wenhui, Shen Nianci: Photodynamic therapy of cancer new drug 3-or 8-3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) deuteroporphyrin IX, Chinese laser medical journal, 1993,2:1; Gu Ying, Liu Fanguang, Wang Kai etc., blood quinoline methyl ether is for the clinical study of photodynamic therapy treatment nevus flammeus, Chinese laser medical journal 2000,9(3): 185).
At present, for the photosensitizers of optical dynamic therapy, major part is complicated mixture, forms uncertainly, structure is still disputable, has affected the stability of medicine preparation, result for the treatment of.Optical dynamic therapy medicine 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) the deuteroporphyrin IX(hemporfin using at present) as single compound, there is clear and definite chemical structure, but still comprise 2 kinds of positional isomerss, its shortcoming having is that character is stable not, thereby has influence on the reliability of effect.The medicine single in order to obtain molecular structure, character is more stable, is necessary further to separate 2 kinds of positional isomerss, for more effectively patient being carried out to optical dynamic therapy.
The fractionation of hemporfin positional isomers is remained to difficult point of the prior art, need to consider a large amount of factors such as the method for splitting that should take, splitting condition (comprising pH value, temperature, time etc.), resolution reagent.In currently available technology, also do not obtain so far such medicine separation, that molecular structure is single and character is more stable.
Summary of the invention
One of object of the present invention is to provide a kind of isomer 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX(hemporfin isomer A of hemporfin), it has the structure shown in formula (I), and this isomer is to adopt the method that comprises the steps from 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX and 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) separate deuteroporphyrin IX mixture:
(1) by 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX and 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX mixture uploads to reverse phase silica gel post;
(2) with the methanol solution that contains damping fluid, pH4～5 are as moving phase wash-out;
(3) detect at 395nm place, collect eluting peak;
(4) wherein, described reverse phase silica gel is selected from: C8 silica gel.
Another object of the present invention is to provide a kind of isomer 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX(hemporfin isomer B of hemporfin), it has the structure shown in formula (II), and this isomer is to adopt the method that comprises the steps from 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX and 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) separate deuteroporphyrin IX mixture:
(1) by 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX and 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX mixture uploads to reverse phase silica gel post;
(2) with the methanol solution that contains damping fluid, pH4～5 are as moving phase wash-out;
(3) detect at 395nm place, collect eluting peak;
Wherein, described reverse phase silica gel is selected from: C8 silica gel.
Said reverse phase silica gel particle diameter is 10～150 μ m, preferable particle size 30～70 μ m; Aperture 60～the 200A of reverse phase silica gel, preferably aperture 60A.
Wherein buffered soln is 1.5M acetic acid--ammonium acetate buffer, and the volume ratio of methyl alcohol and acetic acid--ammonium acetate buffer is 65:35, pH4.76.
Separation system adopts Flash150M, built-in prepackage C
8chromatography column 150mm ID × 30cm, is produced by Biotage company, and silica gel particle diameter is 35～70 μ m, and aperture is 60A;
Preparation of samples: get 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-30 grams of (1-hydroxyethyl) deuteroporphyrin IX sterlings, use tetrahydrofuran (THF) dissolution filter, add 100 grams of C8 reverse phase silica gels, concentrating under reduced pressure, evaporate to dryness, add in loading post SIM, in order to loading;
Loading and collection: so that pH4.76 methyl alcohol-1.5M acetic acid--amine acetate damping fluid is as moving phase, above-mentioned sample is passed through to loading post SIM, bring in chromatography column and separated, flow rate control is at 200ml/min, detector wavelength 395nm, collects hemporfin isomer A flow point 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX and hemporfin isomer B flow point 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX.
Brief description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) deuteroporphyrin IX.
Fig. 2 A is 3-after purifying (1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX sterling HPLC collection of illustrative plates.
Fig. 2 B is 8-after purifying (1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX sterling HPLC collection of illustrative plates.
Fig. 3 A is two kinds of content of isomer of 5 minutes hepatic tissues after administration.
Fig. 3 B is two kinds of content of isomer of 30 minutes hepatic tissues after administration.
Fig. 3 C is two kinds of content of isomer of 8 hours hepatic tissues after administration.
Embodiment
The inventor is through extensive work, from 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) deuteroporphyrin IX, isolate 2 kinds of positional isomerss first, obtain molecular structure single, the medicine that character is more stable.
And, inventor's discovery, the isomer A metabolism of hemporfin is very fast, and liver is accumulated few, and application has lower liver toxicity than mixture separately, can reduce such untoward reaction; And the isomer B of hemporfin is higher in liver concentration, the residence time is long, is more conducive to the optical dynamic therapy effect taking liver as target, as the optical dynamic therapy of liver cancer.
Photoactive compounds
The chemical structure confirmation of two kinds of isomer adopts the poor spectrum of one dimension NOE (1D-NOEDIFF) technology, and result is as follows:
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 aliphatics proton signal; δ-3.98ppm is NH proton signal in ring, and this is because the impact that just shielded by fragrant circulation appears at abnormal High-Field.On the basis of aforementioned hemporfin nuclear magnetic resonance spectroscopy test report, isomer A is carried out to Assignment: 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 the poor spectrum of isomer A one dimension NOE: irradiation δ 10.78, δ 6.54, δ 3.69, δ 2.16 produce NOE gain; Irradiation δ 10.54, δ 6.15, δ 3.64, δ 3.56, δ 2.16 produce NOE gain; Irradiation δ 10.28, δ 4.35, δ 3.22 produce NOE gain; Irradiation δ 10.23, δ 3.73, δ 3.62 produce NOE gain. these digital proof isomer A substituting group CHOHCH
3be connected on C-8 position CH-(OCH
3) CH
3be connected on 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.
Can determine that said isomer A is 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX, for molecular structure is following formula:
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 aliphatics proton signal; δ-3.98ppm is NH proton signal in ring, and this is because the impact that just shielded by fragrant circulation appears at abnormal High-Field.On the basis of aforementioned hemporfin nuclear magnetic resonance spectroscopy test report, the Assignment of isomer B: 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 the poor spectrum of isomer B one dimension: irradiation δ 10.70, δ 6.55, δ 3.64, δ 3.62, δ 3.38 produce NOE gain; Irradiation δ 10.57, δ 6.13, δ 3.71, δ 3.68 produce NOE gain; Irradiation δ 10.28 and δ 10.25, δ 4.35, δ 3.71, δ 3.68, δ 3.64, δ 3.62 produce NOE gain; Irradiation δ 6.55, δ 10.7, δ 3.71, δ 3.68, δ 3.38, δ 2.16 produce NOE gain; Irradiation δ 6.13, δ 10.57, δ 3.71, δ 3.68, δ 3.56, δ 3.53 produce 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 determine that said isomer B is 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX, molecular structure is following formula:
Treatment mechanism
The present invention relates to form the disease causing with optical dynamic therapy because of harmful new vessel, photodynamic therapy scheme makes harmful neovascularization reduction.
In the method for the invention, give and need the patient for the treatment of to take applicable Photoactive compounds, present in an amount at least sufficient to make the Photoactive compounds of patient's intralesional to reach effective concentration.After taking, through after a while, the compound of effective concentration is gathered in after focus, uses this region of rayed being absorbed by this Photoactive compounds.Photoactive compounds, by optical excitation, produces reactive oxygen species and free radicals, 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, as oral, parenterai administration or rectal administration, are advisable, as intravenously, intramuscular or subcutaneous injection with parenterai administration.Taking intravenous injection as best.
The dosage of Photoactive compounds can be according to administering mode, preparation type, whether coupling target part varies widely.It is generally acknowledged, relevant between preparation, administering mode and the dosage level of optical active matter.Conventionally, the exemplary dosage scope of 3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX or 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX is 0.1-100mg/kg, preferably dosage range is 0.5-50mg/kg, and better dosage range is 1-10mg/kg.Those skilled in the art also can be determined by experiment suitable dosage.
To be mutually related for various parameters effective, selective light photodynamic therapy in the present invention.Therefore, can such as, adjust dosage with respect to other parameters (timed interval between optical throughput, illuminance, time length and dosage, administration and the rayed using in photodynamic therapy etc.).The obvious damage that uses these parameters all should adjust to significantly improve eyesight and do not produce normal eyes tissue is advisable.Those skilled in the art can be determined by experiment suitable parameter.
In other words,, in the time that the dosage of Photoactive compounds reduces, the optical throughput of sealing choroidal neovascular tissue has the trend of increase; Otherwise need to reduce optical throughput, need to increase the dosage of Photoactive compounds or increase targeting to promote to increase the enrichment degree of diseased region Photoactive compounds.
Light methods for the treatment of
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, also claims power level, power density.These terms are that those skilled in the art uses and understands, and are illustrated at this.
After Photoactive compounds administration, the target tissue of eye is irradiated under selecteed drug absorption wavelength.For hematoporphyrin monomethyl ether, selected wavelength region, generally in 630 ± 20nm left and right, is more preferably that the penetration power of this range of wavelength in body tissue is relatively good in 630 ± 10nm left and right.
The result of irradiating causes Photoactive compounds in excited state, and with other Compound Phase mutual effects, form singlet oxygen (Singlet Oxygen) and other free radicals, cause the structure deteriorate of blood vessel epithelial cell.Singlet oxygen and other free 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, de-particle and thrombosis, causes 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 large variation.But be preferably 50-200 joule/cm
2.
Illuminance generally changes in 50-800mW/cm
2, with about 100-600mW/cm
2for good.But the illuminance that choice for use is higher, can shorten treatment time and reach same effect.
After Photoactive compounds administration to the optimum time interval between light treatment also according to administering mode, form of medication and preparation type and difference.The timed interval after photosensitizers administration, from 1 minute to 2 hours, is preferably 5-30 minute, and that better is 10-25 minute.
Medicable disease
3-provided by the invention (1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX or 8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX, form the medicine of the disease causing for the preparation for the treatment of because of harmful new vessel, this medicine forms the photosensitizers of the disease causing as optical dynamic therapy because of harmful new vessel.
Because forming the disease causing, harmful new vessel comprises: dermatosis, as nevus flammeus; Also comprise ophthalmic diseases, as macular degeneration.
The evaluation for the treatment of
The effect of optical dynamic therapy is the damage of available histology sections observation endotheliocyte and the encapsulation situations of lesions position new vessel on animal model.Typically, the destruction of new vessel shows as in vascular endothelial cell endochylema and occurs cavity, and nucleus shrinkage is abnormal, the formation of the visible platelet aggregation of Endovascular and blood clot.
Be positioned at the directly observing effect of blood vessel sealing of skin; Be positioned at the blood vessel sealing of organizing deep layer, for example eye choroid, available Angiography 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.Meanwhile, the main untoward reaction of porphyrin medicine is possible cause dysfunction of liver, can show as transaminase and raise.Inventor's discovery, the isomer A metabolism of hemporfin is very fast, and liver is accumulated few, and application has lower liver toxicity than mixture separately, can reduce such untoward reaction.
Meanwhile, the isomer B of hemporfin is higher in liver concentration, and the residence time is long, and prompting is more conducive to the optical dynamic therapy effect taking liver as target, as the optical dynamic therapy of liver cancer.
Therefore,, in the time of concrete enforcement treatment, can carry out according to patient's situation isomer A or the isomer B of choice for use hemporfin, to reach best result for the treatment of.
Major advantage of the present invention is:
(1) provide first a kind of separation 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-(1-hydroxyethyl) deuteroporphyrin IX(hemporfin) method of positional isomers, it is single that the 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 savings is few, and application has lower liver toxicity than mixture separately; And the isomer B of hemporfin is higher in liver concentration, the residence time is long, and prompting is more conducive to the optical dynamic therapy effect taking liver as target.Thereby in the time of concrete enforcement treatment, can carry out according to patient's situation isomer A or the isomer B of choice for use hemporfin.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, the condition of conventionally advising according to normal condition or according to manufacturer.
Embodiment 1: the preparation of hemporfin
The preparation of 1.3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) deuteroporphyrin IX crude product
In the acidproof lass lining reactor of 50L, adding volume ratio is the methanol/water mixed solution 30L of 3:1, under stirring, slowly splashed into by Liquid titrator, the method of recording by Chinese patent 01105208.2 specification sheets embodiment 1 prepare 3,8-bis--(1-bromotrifluoromethane)-deuteroporphyrin IX hydrobromate Glacial acetic acid saturated solution 10L, controls rate of addition and makes reaction mixture temperature remain on 10～20 DEG C.Dropwise, reaction solution continues to stir 2h, places 4h.Then be strong basicity (PH13 left and right) in stirring the lower 10N of the dropping NaOH aqueous solution to reaction solution, more than placing 10h, add acetic acid and be neutralized to PH4～5, add the water dilution of 5 times of volumes, placement is spent the night, throw out natural sedimentation, incline after supernatant liquor, suction strainer collecting precipitation, water fully washs, drain, put and in vacuum drier, use P
2o
5vacuum-drying.Obtain auburn 3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) the deuteroporphyrin IX (53.3%HPLC), 3 that contains, 8-bis--(1-methoxy ethyl)-deuteroporphyrin IX (19.5%HPLC) and 3,160 grams of the crude products of 8-bis--(1-hydroxyethyl)-deuteroporphyrin IX (27.1%HPLC), yield 80%.
The chromatographic separation and purification of 2.3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) deuteroporphyrin IX
Get 10 of 60 × 600mm glass chromatography columns, add respectively the activated silica gel of 200g, carefully strike reality.Get after 3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) deuteroporphyrin IX crude product 6g prepared by previous step and 30g silica gel H grind well and be added on silicagel column upper strata, and separately add silica gel H 20g thereon, ditto strike reality.Add developping agent (methyl alcohol-chloroform-formic acid (10:1:0.1)) and carry out chromatographic separation, Fractional Collections flow point, first detect with thin-layer chromatography, in the time that the required component of TLC detection display is single spot, carry out HPLC detection, merge the flow point that HPLC analyzes relative peak area >=95% of retention time 3.43min, washing, organic phase evaporated under reduced pressure, obtain the about 10g of 3 (8)-(1-methoxy ethyl)-8 (3)-(1-hydroxyethyl) deuteroporphyrin IX (hemporfin) sterling (yield 16.7%).
Embodiment 2: the HPLC of hemporfin positional isomers separates
Instrument: Agilent1100 type high performance liquid chromatograph; DAD detector, detects wavelength: 395nm
Moving phase: methyl alcohol-1.5M acetic acid--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, hemporfin sample prepared by above-described embodiment 1, dissolve with HPLC moving phase, be mixed with the sample solution of 5mg/mL concentration, before sample introduction, filter.Each above-mentioned solution 400 microlitres of sample introduction, after sample introduction, collect respectively flow point A according to HPLC color atlas (seeing Fig. 1), and it is 14.35 points with flow point B retention time that retention time is 11.94 points.Accumulative total 40 times, collects flow point A136ml, flow point B133ml altogether.Collect liquid and pour ethyl acetate (or ether) into--in distilled water two-phase solution, jolting, divides and gets organic phase, with distilled water wash organic phase 3 times, organic phase removes solvent under reduced pressure to dry at 40 DEG C, puts in vacuum drier, spends the night with Vanadium Pentoxide in FLAKES vacuum-drying.
As a result, obtain respectively hemporfin positional isomers A36.2 milligram, isomer B35.0 milligram.
Fig. 2 A is shown in by the HPLC collection of illustrative plates of 3-after purifying (1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX (hemporfin isomer B) sterling.
HPLC collection of illustrative plates Fig. 2 B of 8-after purifying (1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX (hemporfin isomer A) sterling.
Embodiment 3: the preparation scale of hemporfin positional isomers separates
Separation system adopts Flash150M, built-in prepackage C
8chromatography column (150mm ID × 30cm), is produced by Biotage company (U.S.), and silica gel particle diameter is 35～70 μ m, and aperture is 60A.
Preparation of samples: get 3 (or 8)-(1-methoxy ethyl)-8 (or 3)-30 grams of (1-hydroxyethyl) deuteroporphyrin IX sterlings (content >95%) prepared by embodiment 1, use tetrahydrofuran (THF) dissolution filter, add 100 grams of C8 reverse phase silica gels, concentrating under reduced pressure, evaporate to dryness, add in loading post SIM, in order to loading.
Loading and collection: taking methyl alcohol-1.5M acetic acid--amine acetate damping fluid (pH4.76) is (65:35V/V) as moving phase, above-mentioned sample is passed through to loading post SIM, bring in chromatography column and separated, flow rate control is at 200ml/min, detector wavelength 395nm, collects hemporfin isomer-A flow point [8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX] and hemporfin isomer-B flow point [3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX].
Aftertreatment: flow point is collected liquid and is extracted with ethyl acetate, then by organic phase concentrating under reduced pressure, evaporate to dryness, drying under reduced pressure, obtains 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 nuclear magnetic resonance analyser
Solvent: DMSO-d6
Interior mark: TMS
2. resolve
Hemporfin isomer A and isomer B be from chemical structure, is only due to upper two substituting groups in C-3 and the C-8 position difference of exchanging.This research adopts one dimension NOE difference spectrum technology (1D-NOEDIFF) to carry out Structural Identification to these two isomer.
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 aliphatics proton signal; δ-3.98ppm is NH proton signal in ring, and this is because the impact that just shielded by fragrant circulation appears at abnormal 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 to Assignment: 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 poor spectrum of the one dimension NOE of isomer A
Irradiation δ 10.78, δ 6.54, δ 3.69, δ 2.16 produce NOE gain; Irradiation δ 10.54, δ 6.15, δ 3.64, δ 3.56, δ 2.16 produce NOE gain; Irradiation δ 10.28, δ 4.35, δ 3.22 produce NOE gain; Irradiation δ 10.23, δ 3.73, δ 3.62 produce NOE gain.These digital proof isomer A substituting group CH-(OCH
3) CH
3be connected on C-8 position CHOHCH
3be connected on 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 Assignment of isomer B
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. the poor spectrum of isomer B one dimension NOE
Irradiation δ 10.70, δ 6.55, δ 3.64, δ 3.62, δ 3.38 produce NOE gain; Irradiation δ 10.57, δ 6.13, δ 3.71, δ 3.68 produce NOE gain; Irradiation δ 10.28 and δ 10.25, δ 4.35, δ 3.71, δ 3.68, δ 3.64, δ 3.62 produce NOE gain; Irradiation δ 6.55, δ 10.7, δ 3.71, δ 3.68, δ 3.38, δ 2.16 produce NOE gain; Irradiation δ 6.13, δ 10.57, δ 3.71, δ 3.68, δ 3.56, δ 3.53 produce NOE gain.These NOE digital proof isomer B substituting group CHOHCH
3be connected on C-8 position CH-(OCH
3) CH
3be connected on 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)-deuteroporphyrin 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)-deuteroporphyrin IX.
Embodiment 5: hemporfin and positional isomers thereof the Photodynamic therapy test to vascular endothelial cell
Get the mouse pulmonary vascular endothelial cell (RPMVEC that growth conditions is good, be called for short EC, prepare by following literature method: Chen SF, Fei X, Li SH.A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries.Microvas Res, 1995,50:119), with after 0.3% trysinization, blow and beat into single cell suspension, use containing the DMEM nutrient solution of 5% serum cell density is adjusted into 5.0 × 10
4individual/ml, and it is inoculated on 96 porocyte culture plates, every hole adds 200 μ l, is placed in incubator and hatches after 18h, the nutrient solution in the each hole of sucking-off.Hemporfin, isomer A, isomer B prepared by above-described embodiment 1, embodiment 2, three kinds of photosensitizerss are mixed with concentration and are respectively the solution of 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 containing serum, under lucifuge condition, every hole adds 200 μ l, each concentration point 6 holes, incubation time is 4h.Utilize copper vapor laser to irradiate, power density is 20mw/cm
2, the time is 1000 seconds, energy density is 20J/cm
2.It is placed on and in incubator, hatches 24h, then in every hole, adds the MTT (becoming 5mg/ml with PBS solution preparation) of 20 μ l, hatches after 4h in incubator, careful sucking-off nutrient solution and MTT, every hole adds the DMSO of 150 μ l, is placed in incubator and hatches after 12h, vibration 5 minutes before microplate reader is measured, set the parameter of microplate reader, select 595nm as measuring wavelength, 655nm, as with reference to wavelength, measures optical density(OD) (OD) value in each hole, print result, asks its mean value.Experiment in triplicate.
Record after the optical density value in each hole, ask its cell survival rate, formula is as follows:
Cell survival rate (%)=(experimental group OD-background group OD)/(completely blank group OD-background group OD) × 100%
Statistical procedures adopts Stata6.0 software (U.S. Computer Resource Center development), gets the mean value of each concentration point cell survival rate of three experiments and makes fitting of a curve, thereby calculate its IC
50.
Experimental results show that these three kinds of photosensitizerss to EC all without dark toxicity.Under copper vapor laser irradiates, hemporfin isomer-A, hemporfin isomer-B and the hemporfin medium lethal dose (IC to EC
50) be respectively 0.368 μ g/ml, 0.412 μ g/ml, 0.493 μ g/ml.The photodynamic killing effect strength similarity of the Human Umbilical Vein Endothelial Cells that two kinds of isomer of hemporfin and hemporfin mediate.
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 can freely drink water after 10 hours, intravenous injection (iv) 10mg/kg hemporfin, respectively at after administration 5,30 and 480min, by femoral artery sacrificed by exsanguination animal.Get blood plasma 0.5ml, and divide immediately and get hepatic tissue.Take and organize about 0.3g (precise weighing), adding 1.0ml tri-distilled water makes after homogenate, with 3ml acetonitrile precipitation albumen (wherein liver tissue homogenate extract after, sample introduction after dilution), in the centrifugal 5min of 2500rpm, get supernatant liquor 0.5ml, in the centrifugal 10min of 15000rpm, after twice high speed centrifugation, get 160 μ l supernatant liquors in automatic sampling bottle, 20 μ l sample introductions.By two kinds of isomer concentration in HPLC method mensuration tissue.
After rat administration 5,30 and 480min after the HPLC collection of illustrative plates of hepatic tissue sample respectively as shown in Fig. 3 A, Fig. 3 B and Fig. 3 C.As seen from the figure, after administration, in hepatic tissue, the content of isomer B is higher than isomer A, and administration only detects isomer B in hepatic tissue after 8 hours.Therefore two isomer are in the difference that is distributed with of liver, and isomer B is higher in liver abundance, thereby better for Hepatoma therapy effect.
Embodiment 7: hemporfin and positional isomers thereof the light dynamic damage test to cockscomb skin
Choose totally 30 of body weight 0.8～1.2kg, the prosperous chickens of the male Lay in 12～16 weeks ages of week, be divided at random 3 groups.Every treated animal first intramuscular injection fiber crops is protected quiet (0.10～0.12mg/kg) anesthesia, and the hemporfin that is 10mg/ml by concentration under lucifuge condition, isomer A, isomer B photosensitizers are injected into the prosperous chicken vein of Lay by the dosage of 20mg/kg.After fixing cockscomb, mark illumination area, every cockscomb is set 4 irradiated regions, and each irradiated region diameter is 1cm.Select copper vapor laser output mixed light to be exported vertically and irradiated by optical fiber, power density is 100mw/cm
2, irradiation time is 20 minutes, irradiation place does not suitably hide.Before and after irradiation, all use resistance dynamometer to detect output rating, ensure that its fluctuation range is between ± 5%.Control group and each experimental group be color and the surface condition of visual inspection in advance when 3 days and 14 days, recording processing position cockscomb after irradiation all, then cutting organizing of cockscomb illumination area, to be placed in immediately 10% formalin fixing, conventional dehydration, specimens paraffin embedding slices, HE dyeing, light Microscopic observation, record its pathological change.
According to the criteria for classifying of cockscomb skin morphological change after photodynamic action, cockscomb skin target tissue and non-target tissue are divided into gently (L to OPK reaction, light reaction), in (M, middle reaction), heavy (S, sever reaction) three degree.Adopt Χ
2between organizing relatively, setting P < 0.05 is significant difference in inspection.Statistical procedures adopts Stata software.
Three kinds of photosensitizers comparative experimentss that photosensitive damage changes substantially to cockscomb skin target tissue and non-target tissue the results are shown in following table:
Three kinds of photosensitizerss to cockscomb skin photosensitive damage substantially change feature relatively in table 1.
Table 1
The result of the light Microscopic observation of the histopathologic slide of three groups of photosensitizerss is shown: papillary layer capillary network reduces, tube chamber is little, visible new capillary vessel, epidermal area and corium deep layer are without change, reticular layer vasodilation, can produce effective damage effect to target tissue, and all there is comparatively clear and definite selectively acting.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. the isomer 8-of hemporfin (1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX, it has the structure shown in formula (I):
2. the isomer 3-of hemporfin (1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX, it has the structure shown in formula (II):
The purposes of 3.8-(1-methoxy ethyl)-3-(1-hydroxyethyl) deuteroporphyrin IX, is characterized in that, for the preparation for the treatment of the medicine that forms the disease causing because of harmful new vessel.
4. purposes as claimed in claim 3, is characterized in that, it is nevus flammeus that described harmful new vessel forms the disease causing.
5. purposes as claimed in claim 3, is characterized in that, it is macular degeneration that described harmful new vessel forms the disease causing.
The purposes of 6.3-(1-methoxy ethyl)-8-(1-hydroxyethyl) deuteroporphyrin IX, is characterized in that, for the preparation of the medicine of Hepatoma therapy.
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Non-Patent Citations (1)
| Title |
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| 许德余，等.光动力治癌新药血卟啉单甲醚（HMME）的研究.《中国激光医学杂志》.1993,第2卷(第1期),第3-7页. * |
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