CN101712642A - 5 site-sodium thiosulfate substituted elsinochrome derivative and preparation method and application thereof - Google Patents
5 site-sodium thiosulfate substituted elsinochrome derivative and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a 5 site-sodium thiosulfate substituted elsinochrome A derivative and a preparation method and application thereof. In the preparation method, elsinochrome, sodium thiosulfate and a solvent are uniformly mixed away from light till pH value is 10-14; after the reaction is finished, the product is obtained. The derivative has the solubility of 5.1mg/mL in a buffer solution or physiological saline water and meets the requirement on safe transmission in intravenous injection administration and blood; and the preponderant lipotropism (the lipid-water allotting coefficient is about 7) of the derivative is maintained on the premise. A biological experiment indicates that the killing rate of the derivative on the human lung cancer cell (A549) is equivalent to 60 percent of the matrix elsinochrome; by the superhigh photodynamic activity of the elsinochrome, the photodynamic activity of the derivative is sufficient for meeting the clinical requirement. In conclusion, the derivative can be directly used for a clinical photodynamic medicament of intravenous injection without further preparation.
Description
Technical field
The present invention relates to a kind of 5-site-sodium thiosulfate substituted elsinochrome derivative and preparation method thereof and application.
Background technology
Photodynamic therapy has developed into the novel clinical treatment method at kinds of tumors and capillary blood vessel class disease, and photo-dynamical medicine is the bottleneck problem of current restriction photodynamic therapy widespread use.Capillary blood vessel class disease, comprise nevus flammeus, macula retinae sex change etc., the clinical medicine classification all belongs to the common disease category, and sickness rate is all at 3-5 ‰, and the former falls ill more than 90% in face, bring great soul pain (yellow snow flower bud etc. to the patient, photodynamic therapy treatment venule odd-shaped progress, stomatology research, 2007,23,582); The latter is considered to the first cause (Ma Qingmin etc., the Progress in Medication of age-related macular degeneration, Hebei Medical University's journal, 2008,29,470) of the elderly's blinding, and photodynamic therapy is the clinical first-selected therapy of these capillary blood vessel class diseases of treatment at present.
The photo-dynamical medicine of capillary blood vessel class disease needs to be transferred to focus in the intravenous injection input body and by blood circulation, therefore, for guarantee medicine in blood safe transmission, avoid self aggregation bolt match capillary vessel, medicine must possess hydrophilic property; The focus target body of capillary blood vessel class disease is the blood vessel cell, and in order to guarantee affinity and the biological photodynamic activity to the target body cell, medicine must have lipotropy simultaneously.Therefore design has the amphiphatic photosensitizers derivative of " optimization " fat water, under the water-soluble prerequisite that satisfies the clinical administration standard, farthest keep the lipophilic characteristic, be the feasible way (Liu Xin that realizes the photo-dynamical medicine clinical practiceization, Institute of Chemistry, Academia Sinica's Ph D dissertation, 2008,41).
For many years, photodynamic therapy mainly is target with antitumor, therefore, photo-dynamical medicine also mainly is main the evaluation with the anti-tumor function, for example: for the therapeutic efficiency of noumenal tumour, " the phototherapy window " of regulation tumour at 600-900nm, the tissue penetration of this wavelength region light is (3-10 millimeter) deeply, is beneficial to the curative effect of noumenal tumour.Yet the focus degree of depth of capillary blood vessel class disease might cause the injury of healthy tissues this moment on the contrary less than 1 millimeter with the darker long wavelength light of penetration depth! Therefore, research and develop the new strategy that personalized photo-dynamical medicine is the medical treatment of light power at disease feature.At present, the photo-dynamical medicine benzene derivatives of porphyrin unit acid (BPD-MA) that is used for the macula retinae sex change of unique in the world approval listing, popular name Visudyne (Verteporfin), its maximum light absorption wavelength is 690nm, its main light absorbing 5 millimeters of Effective depth penetration of organizing, the existing market price is 16000 yuans/10 milligrams, in addition, the general characteristic of porphyrin class medicine is that metabolism is slow in vivo, and the meta-bolites protoporphyrin still has photosensitive activity, can make photosensitive side effect continue a couple of days and even several months.
(comprise that plain HA of first and second element HB) Tong Shi the perylene naphthoquinone derivatives, EA only is a kind of submember in the wild red bamboo fungus for Elsinochrome A (elsinochrome A, be called for short EA) and hypocrellin.Recently, the artificial bio-membrane synthetic (Li Cong etc., the biosynthetic means of perylene naphtoquinone compounds, patent No. ZL 200510010612.X) of the real perylene quinones phytochrome now of Yunnan University's success, wherein EA is the main component of synthetic, and can realize producing in batches.EA singlet oxygen quantum yield is 0.98 (Li, C., etc, Photophysical and photosensitive propertiesof Elsinochrome A, Chin.Sci.Bull, 2006,51,1050), tumour cell experimental results show that the photodynamic activity of EA is 2～3 times of (Liu of Hypocrellin B, L.H.etc, Killing efect of elsinochrome A andhypocrellin B-mediated photodynamic therapy on ECV304Cells:a comparative study, Chin.J.Laser.Med.Surg, 2006,15,93).In addition, Elsinochrome A can suppress growth (Zhang, the J.etc of gram-positive bacteria, Study on the Bacteriostasis Efect of Elsinochrome A in vitro, Lishizhen.Med.Mater.Medica.Res, 2006,17,2414).
The absorption spectrum and the hypocrellin of Elsinochrome A (EA) are close, as shown in Figure 1, main absorb light (460nm) organize 1 millimeter of Effective depth penetration less than, conform to the focus degree of depth of capillary blood vessel class disease just.The target body of capillary blood vessel class disease is the capillary blood vessel tapetum cell of focal zone, and microvascular disease is that the heteroplasia capillary blood vessel is intensive, and blood fortune is abundant, and medicine can be in the focal zone enrichment behind the intravenous administration; Focal zone contacts with blood flow, and oxygen supply is abundant, and is more responsive to photodynamic therapy.Therefore, photodynamic therapy has now become the method for " the most desirable " of this type of microvascular disease of clinical treatment.
The photo-dynamical medicine of capillary blood vessel class disease need be transported to focal zone by blood circulation, and the Elsinochrome element strong lipotropy organic molecule that is a class, and water-soluble extremely low (mcg/ml) can spontaneous gathering in blood and cause capillary embolism! The plain water-soluble transformation of Elsinochrome is the effective means that solves medicine practicability problem.
The Elsinochrome element is that hypocrellin belongs to homologue, and structural difference is that the seven-membered ring of hypocrellin is 6 yuan of rings in the Elsinochrome element, have symmetric chemical structure.Experiment records, and the solubleness of Elsinochrome A is 1.4 μ g/mL, and (4.6 μ g/mL) more is insoluble in water than hypocrellin; Early-stage Study shows that Elsinochrome A is difficult to prepare Liposomal formulation, seek that chemical modification method improves the Elsinochrome element water-soluble be the possible approach that realizes medicine practicability.Compare with hypocrellin, the chemically modified work of Elsinochrome element is considerably less, report only has the room temperature synthetic bromide for a kind of (Huang of Elsinochrome A at present, S., etc, The bromination of elsinochrome A andphotosensitivity of it s derivatives, J.Yunnan Univ, 2003,25,352).Yet bromo derivative can not effectively improve the wetting ability of Elsinochrome element, and bromine may be introduced toxicity.Thermochemical method is adopted in the chemically modified of hypocrellin mostly, the reaction system top temperature can reach 140 ℃ of (Song, Y.Z., ESR studies ofthe photodynamic properties of a long-wavelength and water-soluble hypocrellin Bderivative:photogeneration of semiquinone radical anion and activated oxygen, J.Photochem.Photobiol.A, 1999,123,39).Inventor's previous experiments is found, plain most of decompose (thin layer chromatography and the mass spectrum inspection) of Elsinochrome when temperature surpasses 50 ℃, when temperature of reaction system surpassed 35 ℃, Elsinochrome A just began to decompose, and produced the compound (result does not deliver) of small molecular weight.
In earlier stage " optimization " or " quantitatively " fat water that proposes according to the inventor notion (Liu Xin of dissolubility that holds concurrently, Institute of Chemistry, Academia Sinica's Ph D dissertation, 2008), be at utmost to keep lipotropy under the water-soluble prerequisite that satisfies clinical photo-dynamical medicine concentration, to guarantee cellular uptake rate and biological light dynamic efficiency! We utilize molecular polarity theoretical prediction method (Xie, J., etc, Prediction on amphiphilicity of hypocrellin derivatives, Sci.China, 2002,45,251), designed, synthesized the Elsinochrome A analog derivative that 3-sulfydryl-1-sulfonic acid replaces.The seven-membered ring of hypocrellin destroyed whole molecule to taking advantage of character, therefore, the derivative of 5,8 replacements of hypocrellin is different compounds; There is C in the Elsinochrome A molecule
2Operation symmetry (shown in formula III), therefore, the Elsinochrome A derivative of 5 or 8 replacements is same compounds, this is the synthetic convenience that provides of analog derivative for this reason.
Summary of the invention
The purpose of this invention is to provide a kind of 5-site-sodium thiosulfate substituted elsinochrome derivative and preparation method thereof and application.
5-site-sodium thiosulfate substituted elsinochrome derivative provided by the invention, its general structure is suc as formula shown in the I:
In the above-mentioned formula I general structure, R is S (CH
2)
nSO
3H, 2≤n≤6, preferred n is 3.
The method of 5-site-sodium thiosulfate substituted elsinochrome derivative of preparation provided by the invention is under the condition of pH value for 10-14 and lucifuge, with Elsinochrome element, formula II general structure (HS (CH
2)
nSO
3H) sulfydryl sulfonic acid shown in and solvent mixing, reaction finishes and obtains 5-site-sodium thiosulfate substituted elsinochrome derivative provided by the invention.
The Elsinochrome element is Elsinochrome A (its structural formula is shown in formula III), and sulfydryl sulfonic acid is 2-sulfydryl-ethyl sulfonic acid, 3-sulfydryl-1-propanesulfonic acid, 4-sulfydryl-1-fourth sulfonic acid, 5-sulfydryl-1-penta sulfonic acid or the own sulfonic acid of 6-sulfydryl-1-; The mol ratio of sulfydryl sulfonic acid and Elsinochrome element 〉=10: 1, preferred 10-20: 1.Solvent is the mixed solution of component A and B component; Wherein, component A is dimethyl sulfoxide (DMSO), N, dinethylformamide, tetrahydrofuran (THF), pyridine, hexanaphthene or dioxane, and B component is the aqueous solution of triethylamine, sodium hydroxide or the aqueous solution of tetramethyl-aqua ammonia.
In this reaction, the pH value of reaction system is 11-14.Temperature of reaction is≤35 ℃, preferred 15-35 ℃; Reaction times is 〉=2 hours, preferred 2-10 hour.After reaction finishes, can do following separation and purification to this reaction system: add the water of reaction system 50 times (volume ratios), adding acid makes the sulfonic acid anion derivative is become sulfonic acid.Use dichloromethane extraction repeatedly, extract organic phase; Distillation under vacuum or rotary evaporation are removed organic solvent; The solid residue dissolve with methanol, with methylene dichloride: methyl alcohol (volume ratio=5: 1) or ethyl acetate: methyl alcohol (volume ratio=3: 1) is developping solution, with silica gel is that filled column chromatography or thin-layer chromatography separate, purifying, obtains the plain derivative of Elsinochrome that 5-sulfydryl sulfonic acid replaces.
Among the above-mentioned preparation method, the reaction mechanism of Elsinochrome A and sulfhydryl compound is 5 of sulfydryl negative ion attack Elsinochrome A, belongs to Michael type nucleophilic addition.Basic solvent (pH value 〉=10) makes 3-sulfydryl-1-propanesulfonic acid form the sulfydryl negative ion, and the Michael reaction is carried out.The mol ratio of Elsinochrome A and 3-sulfydryl-1-propanesulfonic acid began to take place more than or equal to reaction in 10: 1, to 20: 1 o'clock productive rate maximums; Temperature is to prevent that Elsinochrome A from producing thermolysis smaller or equal to 35 ℃; Reaction times to 4 hour derivative productive rate maximum; Lucifuge is in order to prevent that Elsinochrome A from producing from quick photoxidation.Utilize derivative in the fat big characteristics that distribute in mutually, extraction repeatedly makes derivative all enter separation and purification again after the organic phase.
The present invention is to realize that the plain clinical practice type of Elsinochrome intravenous injection injection is a target, and the Elsinochrome A derivative that provides a kind of 5-sulfydryl sulfonic acid to replace reaches method and the application thereof at following this derivative of chemosynthesis of room temperature.This derivative solubleness in buffered soln or physiological saline reaches 5.1mg/mL, satisfies the requirement of safe transmission in intravenous administration and the blood; Put the advantage lipotropy (lipid is about 7) that has kept derivative before this.Bioexperiment proves that this derivative is equivalent to 60% of parent Elsinochrome element to the kill rate of human lung carcinoma cell (A549); In view of the photodynamic activity of the plain superelevation of Elsinochrome, the photodynamic activity of this derivative is enough to satisfy clinical requirement.Conclusion: this derivative further preparation, can be directly used in intravenous clinical photo-dynamical medicine.
Description of drawings
Fig. 1 is the absorption spectrum of EA and SEA.
Fig. 2 is the semiquinone negatively charged ion that produces by the SEA that ESR detects.
Fig. 3 be the creating singlet oxygen by using that produces by the SEA that ESR detects (
1O
2) signal.
Fig. 4 is the superoxide anion free radical (O that produces by the SEA that ESR detects
2 -) signal.
Fig. 5 is hydroxyl radical free radical (OH) signal that produces by the SEA that ESR detects.
Fig. 6 is for passing through 9, the creating singlet oxygen by using quantum yield with light activated SEA of 470-900nm and EA that the 10-DPA bleaching process detects.
Fig. 7 is the kill rates of three kinds of photosensitizerss to the A549 cell.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
50 milligram (0.92 * 10 of Elsinochrome A (EA)
-4Mole), 250 milligrams 3-sulfydryl-1-propanesulfonic acid sodium (1.6 * 10
-3Mole) and 50 ml volumes ratio be 1: 1 dimethyl sulfoxide (DMSO) and triethylamine, pH=11 puts into 100 milliliter of three neck round-bottomed flask, normal temperature induction stirring 4 hours.Use dichloromethane extraction after the washing repeatedly, add hydrochloric acid and make derivative enter organic phase from water.An amount of dissolve with methanol of pressure reducing and steaming organic solvent, residuum separates with the silica-gel plate thin-layer chromatography, and developping solution is a methylene dichloride: methyl alcohol, volume ratio=5: 1.Collect the red-brown product component at Rf value 0.52 place, further use this method chromatography purification, obtain the Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid replaces, productive rate 40%.
Utilize methods such as spectrum, nuclear-magnetism, mass spectrum that this compound is carried out composition, structural analysis, the result is as follows:
UV spectrum λ
Max: 482nm, 580nm;
Infrared spectra v
Max: 1610cm
-1, 1710cm
-1, 2936cm
-1, 3406cm
-1
Nucleus magnetic resonance
δ(
1H)：6.91(s)，5.19(d)，4.19(s)，3.67(s)，2.14(s)，1.17(s)ppm；
Mass spectroscopy (ESI): 697 (M-1)
-
50 milligram (0.92 * 10 of Elsinochrome A (EA)
-4Mole), 500 milligrams 3-sulfydryl-1-propanesulfonic acid sodium (3.2 * 10
-3Mole) and 60 ml volumes ratio be 2: 1 N, dinethylformamide and be 25% the tetramethyl-aqua ammonia aqueous solution, pH=14 puts into 100 milliliter of three neck round-bottomed flask, normal temperature induction stirring 8 hours.Use dichloromethane extraction after the washing repeatedly, add hydrochloric acid and make derivative enter organic phase from water.An amount of dissolve with methanol of pressure reducing and steaming organic solvent, residuum separates with the silica-gel plate thin-layer chromatography, and developping solution is a methylene dichloride: methyl alcohol, volume ratio=5: 1.Collect the red-brown product component at Rf value 0.52 place, further use this method chromatography purification, obtain the Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid replaces, productive rate 28%.
Utilize methods such as spectrum, nuclear-magnetism, mass spectrum that this compound is carried out composition, structural analysis, the result is as follows:
UV spectrum λ
Max: 482nm, 580nm;
Infrared spectra v
Max: 1610cm
-1, 1710cm
-1, 2936cm
-1, 3406cm
-1
Nucleus magnetic resonance
δ(
1H)：6.91(s)，5.19(d)，4.19(s)，3.67(s)，2.14(s)，1.17(s)ppm；
Mass spectroscopy (ESI): 697 (M-1)
-
50 milligram (0.92 * 10 of Elsinochrome A (EA)
-4Mole), 250 milligrams 3-sulfydryl-1-propanesulfonic acid sodium (1.6 * 10
-3Mole) and 30 milliliters of adjusting pH values be 10 N, dinethylformamide and aqueous sodium hydroxide solution, (N, the volume ratio of dinethylformamide and sodium hydroxide is 1: 1,8% aqueous sodium hydroxide solution), puts into 100 milliliter of three neck round-bottomed flask, normal temperature induction stirring 10 hours.Use dichloromethane extraction after the washing repeatedly, add acetic acid and make derivative enter organic phase from water.An amount of dissolve with methanol of pressure reducing and steaming organic solvent, residuum separates with the silica-gel plate thin-layer chromatography, and developping solution is a methylene dichloride: methyl alcohol, volume ratio=5: 1.Collect the red-brown product component at Rf value 0.52 place, further use this method chromatography purification, obtain the Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid replaces, productive rate 23%.
Utilize methods such as spectrum, nuclear-magnetism, mass spectrum that this compound is carried out composition, structural analysis, the result is as follows:
UV spectrum λ
Max: 482nm, 580nm;
Infrared spectra v
Max: 1610cm
-1, 1710cm
-1, 2936cm
-1, 3406cm
-1
Nucleus magnetic resonance
δ(
1H)：6.91(s)，5.19(d)，4.19(s)，3.67(s)，2.14(s)，1.17(s)ppm；
Mass spectroscopy (ESI): 697 (M-1)
-
100 milligram (1.84 * 10 of Elsinochrome A (EA)
-4Mole), 500 milligrams 3-sulfydryl-1-propanesulfonic acid sodium (3.2 * 10
-3Mole) and 60 ml volumes ratio be 1: 1 dimethyl sulfoxide (DMSO) and aqueous sodium hydroxide solution (2M), pH=14 puts into 100 milliliter of three neck round-bottomed flask, normal temperature induction stirring 4 hours.Use chloroform extraction after the washing repeatedly, add acetic acid and make derivative enter organic phase from water.An amount of dissolve with methanol of pressure reducing and steaming organic solvent, residuum separates with silicagel column, and elutriant is an ethyl acetate: methyl alcohol, volume ratio=5: 1.Collect red-brown product component, further use silica-gel plate thin-layer chromatography purifying, developping solution is a methylene dichloride: methyl alcohol=5: 1 obtains the Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid replaces, productive rate 17%.
Utilize methods such as spectrum, nuclear-magnetism, mass spectrum that this compound is carried out composition, structural analysis, the result is as follows:
UV spectrum λ
Max: 482nm, 580nm;
Infrared spectra v
Max: 1610cm
-1, 1710cm
-1, 2936cm
-1, 3406cm
-1
Nucleus magnetic resonance
δ(
1H)：6.91(s)，5.19(d)，4.19(s)，3.67(s)，2.14(s)，1.17(s)ppm；
Mass spectroscopy (ESI): 697 (M-1)
-
100 milligram (1.84 * 10 of Elsinochrome A (EA)
-4Mole), the 3-sulfydryl-1-propanesulfonic acid sodium (9.28 * 10 of 1.45 grams
-3Mole) and 60 ml volumes ratio be 1: 1 the dimethyl sulfoxide (DMSO) and the 25% tetramethyl-aqua ammonia aqueous solution, pH=14 puts into 100 milliliter of three neck round-bottomed flask, normal temperature induction stirring 7 hours.Use chloroform extraction after washing with water repeatedly, add hydrochloric acid and make derivative enter organic phase from water.Pressure reducing and steaming organic solvent, residuum separate with the silica-gel plate thin-layer chromatography after with dissolve with methanol, and developping solution is an ethyl acetate: methyl alcohol, volume ratio=3: 1.Collect the red-brown product component at Rf value 0.3 place, further use this method chromatography purification, obtain the Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid replaces, productive rate 21%.
Utilize methods such as spectrum, nuclear-magnetism, mass spectrum that this compound is carried out composition, structural analysis, the result is as follows:
UV spectrum λ
Max: 482nm, 580nm;
Infrared spectra v
Max: 1610cm
-1, 1710cm
-1, 2936cm
-1, 3406cm
-1
Nucleus magnetic resonance
δ(
1H)：6.91(s)，5.19(d)，4.19(s)，3.67(s)，2.14(s)，1.17(s)ppm；
Mass spectroscopy (ESI): 697 (M-1)
-
Embodiment 6,5-site-sodium thiosulfate substituted elsinochrome derivative of preparation
50 milligram (0.92 * 10 of Elsinochrome A (EA)
-4Mole), 500 milligrams 3-sulfydryl-1-propanesulfonic acid sodium (3.2 * 10
-3The mole) and 30 ml volumes ratio be that 1: 1 the pyridine and 25% the tetramethyl-aqua ammonia aqueous solution are put into 50 milliliter of three neck round-bottomed flask, heat 100 ℃, mechanical stirring 8 hours.Use chloroform extraction after the washing repeatedly, add acetic acid and make derivative enter organic phase from water.An amount of dissolve with methanol of pressure reducing and steaming organic solvent, residuum separates with silicagel column, and elutriant is an ethyl acetate: methyl alcohol, volume ratio=5: 1.Collect red-brown product component, further use silica-gel plate thin-layer chromatography purifying, developping solution is a methylene dichloride: methyl alcohol=5: 1 obtains the Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid replaces, productive rate 15%.
Utilize methods such as spectrum, nuclear-magnetism, mass spectrum that this compound is carried out composition, structural analysis, the result is as follows:
UV spectrum λ
Max: 482nm, 580nm;
Infrared spectra v
Max: 1610cm
-1, 1710cm
-1, 2936cm
-1, 3406cm
-1
Nucleus magnetic resonance
δ(
1H)：6.91(s)，5.19(d)，4.19(s)，3.67(s)，2.14(s)，1.17(s)ppm；
Mass spectroscopy (ESI): 697 (M-1)
-
The amphiphilic experiment of fat water of 7,5-site-sodium thiosulfate substituted elsinochrome derivative of embodiment
Fat moisture proportioning test carries out in n-Octanol and damping fluid (PBS) system: the 2mg photosensitizers is joined in 2 milliliters of n-Octanols, add 2 milliliters of PBS (pH=7.4) then.Mixing solutions was placed ultrasonic wave vibration 20 minutes, under the speed that per minute 4000 changes centrifugal 10 minutes then, make two to be separated.Measure the absorption spectrum of photosensitizers in the two-phase.
According to the fat water dispenser of Lambert-beer law photosensitizers than (PC) following calculating: PC=ε
PBSA
OCT/ ε
OCTA
PBS,
Wherein, ε
PBSAnd ε
OCTBe the molar extinction coefficient of photosensitizers in PBS and n-Octanol; A
PBSAnd A
OCTBe the obtained the maximum absorption of photosensitizers in PBS and n-Octanol.
The Elsinochrome A derivative that 5-3-sulfydryl-1-propanesulfonic acid is replaced is at n-Octanol and phosphate buffered saline buffer (PBS according to the method described above, pH=7.4) in lipid and in PBS (pH=7.4) maxima solubility measure, its result all lists in the table 1.
The amphiphilic evaluation table of Elsinochrome A derivative fat water that table 15-3-sulfydryl-1-propanesulfonic acid replaces
Wherein, SEA is the Elsinochrome A derivative that 5-3-sulfydryl provided by the invention-1-propanesulfonic acid replaces; EA is the parent Elsinochrome A, and HB is a Hypocrellin B.As known from Table 1, SEA solubleness reaches 5mg/mL, satisfies the clinical medicine concentration requirement; Its fat water dispenser has kept the fat of advantage to distribute mutually than near 7.Cellular uptake rate and photosensitization activity are two prerequisites of the biological photodynamic activity of photosensitizers, and the fat of advantage distributes mutually and characterizes the lipotropy feature of photosensitizers, is the prerequisite of cellular uptake.
8,5-3-of embodiment sulfydryl-1-propanesulfonic acid replaces the photosensitization activity experiment of Elsinochrome A derivative
The DMSO solution that 5-3-of the 1mM sulfydryl that the illumination argon gas is saturated-1-propanesulfonic acid replaces Elsinochrome A can produce an epr signal (g=2.0051) (as Fig. 2), the semiquinone radical anion signal similar that signal and HB produce, this signal ownership is the semiquinone radical anion signal that 5-3-sulfydryl-1-propanesulfonic acid replaces Elsinochrome A; TEMP spin trapping creating singlet oxygen by using quantitatively forms TEMPO, and the latter can be detected by EPR.5-3-of the 1mM sulfydryl of illumination oxygenation-1-propanesulfonic acid replaces the DMSO solution of Elsinochrome A and 50mM TEMP, can observe an equicohesive triplet epr signal (as Fig. 3), its g-factor and split branch constant (g=2.0056, α
N=16.3G); Ultra-oxygen anion free radical is caught the back by DMPO and is detected by EPR, and as shown in Figure 4, the 5-3-sulfydryl of illumination oxygenation-1-propanesulfonic acid replaces the DMSO solution of Elsinochrome A and 50mM DMPO, and the parameter that produces the free radical signal is g=2.0056, α
N=13.0G, α
β H=10.3G and α
γH=1.5G, these parameters and DMPO-O
2 -Adducts is identical; When 5-3-of the 1mM sulfydryl of illumination oxygenation-1-propanesulfonic acid replaces PBS (pH=7.4) solution of Elsinochrome A and 50mM DMPO, can observe the signal (α of DMPO-OH adducts
N=α γ
H=14.9G, the peak is by force than being 1: 2: 2: 1) as shown in Figure 5; The creating singlet oxygen by using quantum yield of sample is measured (as Fig. 6) with the DPA bleaching method, and light source is a pressure sodium lamp among the 450W, clips light less than 470nm with spectral filter.Each compound is adjusted to unanimity at the integral area of 470-800nm optical density value.
5-3-of synthetic of the present invention sulfydryl-1-propanesulfonic acid replaces the Elsinochrome A derivative can photosensitization produce the semiquinone negatively charged ion,
1O
2, O
2 -With the OH free radical, as Fig. 2-shown in Figure 6.As can be known, 5-3-sulfydryl provided by the invention-1-propanesulfonic acid replace the Elsinochrome A derivative can effectively produce semiquinone negatively charged ion (as shown in Figure 2), creating singlet oxygen by using (
1O
2) (as shown in Figure 3), superoxide anion free radical (O
2 -) (as shown in Figure 4), hydroxyl radical free radical (OH) (as shown in Figure 5), the creating singlet oxygen by using quantum yield is that 0.53 (derivative transfers under the condition of equating in the 470-800nm absorbing amount with parent and excites, as shown in Figure 6).
9,5-3-of embodiment sulfydryl-1-propanesulfonic acid replaces the biological photodynamic activity experiment of Elsinochrome A derivative
5-3-sulfydryl-1-propanesulfonic acid replaces Elsinochrome A derivative photodynamic activity and characterizes with the cell experiment result: the human lung carcinoma cell of vitro culture (A549), and by 5.0 * 10
4The cell density of individual/ml is inoculated in 96 porocyte culture plates, hatch 24 hours after, add photosensitizers, continue to hatch lung carcinoma cell after 4 hours, adopting wavelength is the 532nm laser radiation, energy density is 20J/cm
2(power density is 20mw/cm
2), irradiation time is 1000 seconds, adopts tetrazolium salts colorimetric test (mtt assay) thereafter, measures the optical density value in each hole, calculates the cell killing rate.All tests are all finished under the condition of aseptic and lucifuge in Bechtop.Wherein, each photosensitizers is got same concentrations point (0.18 μ M is in the pH value is 7.4 PBS solution) and is measured, and utilizes following formula to ask its cell killing rate:
Cell killing rate (%)=1-cell survival rate
The gained kill rate is the result all list in the table 2.
The kill rate of the Elsinochrome A derivative pair cell that table 25-3-sulfydryl-1-propanesulfonic acid replaces
Cell experiment shows, the cell killing rate of the Elsinochrome A (SEA) that 5-3-sulfydryl-1-propanesulfonic acid replaces be parent Elsinochrome A (EA) 60% and Hypocrellin B (HB) 70%, as shown in Figure 7.Because Elsinochrome A and the high characteristics of hypocrellin photodynamic activity, this derivative has kept most of photodynamic activity of parent, be enough to become a kind of highly active clinical photo-dynamical medicine, and owing to water-solublely satisfy clinical application concentration, need not preparation, can prepare intravenous (IV) drug easily.
Claims (10)
1. 5-site-sodium thiosulfate substituted elsinochrome derivative, its general structure is suc as formula shown in the I:
Wherein, in the described formula I general structure, R is S (CH
2)
nSO
3H, 2≤n≤6.
2. derivative according to claim 1 is characterized in that: in the described formula I general structure, n is 3.
3. method for preparing claim 1 or 2 described 5-site-sodium thiosulfate substituted elsinochrome derivatives, be under the condition of pH value for 10-14 and lucifuge, with sulfydryl sulfonic acid and the solvent mixing shown in Elsinochrome element, the formula II general structure, reaction finishes and obtains described 5-site-sodium thiosulfate substituted elsinochrome derivative; Wherein, in the described formula II general structure, 2≤n≤6.
HS (CH
2)
nSO
3H formula II
4. method according to claim 3 is characterized in that: described Elsinochrome is plain to be Elsinochrome A; Described sulfydryl sulfonic acid is 2-sulfydryl-ethyl sulfonic acid, 3-sulfydryl-1-propanesulfonic acid, 4-sulfydryl-1-fourth sulfonic acid, 5-sulfydryl-1-penta sulfonic acid or the own sulfonic acid of 6-sulfydryl-1-.
5. method according to claim 4 is characterized in that: described sulfydryl sulfonic acid is 3-sulfydryl-1-propanesulfonic acid.
6. according to the arbitrary described method of claim 3-5, it is characterized in that: the mol ratio of described sulfydryl sulfonic acid and Elsinochrome element 〉=10: 1, preferred 10-20: 1.
7. according to the arbitrary described method of claim 3-5, it is characterized in that: described temperature of reaction is≤35 ℃, preferred 15-35 ℃; Reaction times is 〉=2 hours, preferred 2-10 hour, and more preferably 4 hours.
8. according to the arbitrary described method of claim 3-5, it is characterized in that: described pH value is 11-14.
9. according to the arbitrary described method of claim 3-5, it is characterized in that: described solvent is the mixed solution of component A and B component;
Wherein, described component A is dimethyl sulfoxide (DMSO), N, dinethylformamide, tetrahydrofuran (THF), pyridine, hexanaphthene or dioxane, and described B component is the aqueous solution of triethylamine, sodium hydroxide or the aqueous solution of tetramethyl-aqua ammonia;
The volume ratio of described component A and B component is 1-10: 1-10;
In the described B component, the mass percent concentration of the aqueous solution of sodium hydroxide is 4-20%, and the mass percent concentration of the aqueous solution of described Tetramethylammonium hydroxide is 4-40%.
10. the photo-dynamical medicine that is activeconstituents with claim 1 or 2 described 5-site-sodium thiosulfate substituted elsinochrome derivatives.
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