CN102174083A - Compositae cyclopeptide, immunosuppressive medicine using compositae cyclopeptide as active ingredient and preparation method and application of compositae cyclopeptide - Google Patents
Compositae cyclopeptide, immunosuppressive medicine using compositae cyclopeptide as active ingredient and preparation method and application of compositae cyclopeptide Download PDFInfo
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- CN102174083A CN102174083A CN2011100390634A CN201110039063A CN102174083A CN 102174083 A CN102174083 A CN 102174083A CN 2011100390634 A CN2011100390634 A CN 2011100390634A CN 201110039063 A CN201110039063 A CN 201110039063A CN 102174083 A CN102174083 A CN 102174083A
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Images
Abstract
Composite family type cyclic peptide compounds refer to that skeleton contains β-phenylalanine, and are connected directly the equal pentapeptide compound of the monocycle to be formed with normal amino acid peptide bond; Typically contain a L- β-phenylalanine, a Serine, a L-PROLINE derivative and two other amino acid derivativges. Wherein structural formula is the composite family type cyclic peptide of (1-5), is the immunosuppressor in novel plant source. The preparation method of such compound is provided and is preparing the application in immunosuppressive drug.
R1, R2=aryl or alkyl, R3, R4, R5=hydroxyl or halogen atom, X, Y=unsaturated bond or saturated bond.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, relate to a class composite family type cyclic peptide particularly, as immunosuppressor, is the pharmaceutical composition of effective constituent with it, its preparation method and the application in the preparation immunosuppressive drug thereof.
Background technology
Immunomodulatory is that body is kept homeostatic key factor.If the function of immune system imbalance, body produces excessive immune response to autoantigen and causes tissue injury, will cause autoimmune disorder or anaphylaxis etc.Immunosuppressor is meant can be by suppressing cell and humoral immune reaction, the class material that tissue injury is alleviated.It is inhibited to the immune response of body, can suppress propagation and function with immune response cells involved (scavenger cells such as T cell and B cell), reduces organism immune response.Immunosuppressor is widely used in the anti-rejection of organ transplantation and autoimmune disease such as rheumatoid arthritis, lupus erythematosus, tinea, film kidney ball ephritis, inflammatory bowel and autoimmune hemolytic anemia etc. at present.At present existing many immunosuppressor are found, mainly be divided into chemical synthesis preparation, hormone, microbial metabolites, Chinese medicine and effective constituent thereof and biological technology products (cytokine, albumen, antibody) etc., the listing of a lot of successful medicines is wherein also arranged and produce huge economic benefit, as derive from S-Neoral, the chemicals endoxan of microbial metabolites, the prednisone of hormones etc.But in the existing immunosuppressive drug, there are shortcomings such as poor specificity, toxic side effect is obvious, the treatment window is narrow and cost an arm and a leg, so the anxious immunosuppression new drug of waiting to develop high-efficiency low-toxicity more.Chinese medicine is the rarity of the Chinese nation, the Chinese medicine immunosuppressor because of composition variation, pharmacological action extensively, advantages such as few side effects, no dependence, now become the main flow in market together with neotype immunosuppressant.So find that from motherland's traditional Chinese medicine material neotype immunosuppressant has crucial academic significance and good application prospects.
The T lymphocyte accounts for leading role in the pathologic process of various autoimmune diseases and transplant rejection etc., suppressor T cell is one of main mechanism of playing a role of immunosuppressive drug.Most of T cells are experiencing the destiny of tranquillization, activation, effect, apoptosis in the immunne response process.Sophisticated T cells is activated by its antigen receptor after antigen recognition, the activated T cell carries out clonal expansion with exponential speed, next be divided into the effector cell, secrete cytokines or direct killing target cell enter apoptosis at last to keep the homeostasis of T cell population.Immunosuppressor cyclosporin A (the Cyclosporin A of relative low toxicity, CsA) can optionally act on the t cell activation stage, stop its activation and proliferation, be widely used in suppressing the rejection that organ transplantation causes clinically, but unsatisfactory to the curative effect of autoimmune disease.This be likely because they to activatory effector T cell restraining effect is not strong, the pathologic reaction that unable prevention has started is to the damage of autologous tissue.Many researchs show also in the pathogenic process of autoimmune disorder that autoreactive T cell is in the continuous activation state that stimulated by autoantigen, constantly discharges inflammatory cytokine, and can assist the B cell to produce a large amount of autoantibodies.In order to suppress ongoing pathologic immunne response, the activated T cell is the good targets of immunosuppressive drug selectively acting.
Do not see in the prior art report of composite family type cyclic peptide as immunosuppressor arranged.
Composite family type cyclic peptide exists kind few in feverfew, only separates obtaining 9 at present from composite family Aster aster.Cyclic peptide separates because of reasons such as the bad content of its solvability is lower are difficult.In the prior art, the separating ring peptide constituents has been published following method from aster: aster dry root methanol extraction, n-butanol extraction gets propyl carbinol and two positions of water.N-butanol portion is through Diaion HP-20 column chromatography, (1:0-0:1) methanol is an eluent, 80%(methanol wherein) component is through silica gel column chromatography, (1:0-0:1) methylene chloride is an eluent, wherein 10% (ethanol/methylene) component is through the separation and purification of ODS HPLC semipreparative column, acetonitrile/water is an eluent, obtains compd A stin C.Referring to: Morita, H.; Nagashima, S.; Takeya, K.; Itokawa, H.; Iitaka, Y., Structures and conformation of antitumour cyclic pentapeptides, astins A, B and C, from
Aster tataricus.
Tetrahedron1995,
51(4), 1121-1132.Its shortcoming is repeatedly to use the mixed solvent system of organic solvent and water in the separation and purification, and this mixed solvent recovered temperature height is unfavorable for the recovery of solvent; All solvents cost an arm and a leg in the HPLC method, make extraction cost too high, and the HPLC technology also is unfavorable for industrial mass production simultaneously.
Summary of the invention
At prior art above shortcomings part, the object of the present invention is to provide a class composite family type cyclic peptide compounds; The method of this compounds of preparation is provided, this extraction and separation method controllability and favorable reproducibility, sample loss is few, and cost is lower, and is easy to operate, can more easily separatedly obtain cyclic peptide, and solvent can be recycled repeatedly, also is applicable to industrial production.The present invention also aims to provide with composite family type cyclic peptide compounds is the pharmaceutical composition of effective constituent; The application in the preparation immunosuppressive drug of this compounds and composition thereof.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Composite family type cyclic peptide compounds is that a class skeleton contains
β-phenylalanine, and with the directly continuous equal pentapeptide compound of monocycle that forms of normal amino acid peptide bond; Generally contain a L
-β-phenylalanine, a L-Serine, a L-proline derivative and two other amino acid derivative.
Cyclic peptide of the present invention, the compd A stin C(1 that preferably has following structural formula), Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))).
R
1, R
2=aryl or alkyl
R
3, R
4, R
5=hydroxyl or halogen atom
X, Y=unsaturated link(age) or saturated bond
The method for preparing the above-mentioned composite family type of the present invention cyclic peptide compounds is got composite family aster rhizome, after drying, the pulverizing, fully extracts with methanol eddy; After methyl alcohol medicinal extract added the water suspendible, fully extract with ethyl acetate and propyl carbinol successively; Ethyl acetate part and propyl carbinol part are used silica gel, the various chromatography column separation and purification of Sephadex LH-20, RP-18 repeatedly, and the TLC detection method in conjunction with cyclic peptide promptly obtains composite family type cyclic peptide again.
Be prepared as follows the type of composite family shown in structural formula cyclic peptide compounds Astin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) method, get the aster rhizome, after drying, the powder essence, extract 3 times with methanol eddy, time is each 1-3 hour, and extracting solution gets methyl alcohol medicinal extract through concentrating under reduced pressure; After methyl alcohol medicinal extract added the water suspendible, fully extract with ethyl acetate and propyl carbinol successively, equal-volume respectively extracts three times, reclaims solvent and obtains ethyl acetate part, propyl carbinol part and water section; Ethyl acetate part through silica gel column chromatography, use 100:0,9:1,8:2,7:3, the chloroform/methanol gradient elution of 0:100, thin-layer chromatography is in conjunction with cyclic peptide TLC detection method merging 9:1 and the 8:1 part that contains cyclic peptide wherein; Each following step all must be carried out separation and purification in conjunction with cyclic peptide TLC detection method; Medicinal extract after the merging is used 20:1 once more through silica gel column chromatography, 10:1, and 85:15,8:2, the chloroform/methanol gradient elution of 0:1 is merged into eight component Fr.1-Fr.8 according to the difference of cyclic peptide point; The Fr.2 component is through silica gel column chromatography, and the sherwood oil of 2:1-1:2/acetone gradient elution merges and obtains six subfraction Fr.2-1 – Fr.2-6; Fr.2-5 is again through silica gel column chromatography, 20:1, and 15:1,10:1, the chloroform/methanol gradient elution of 5:1 merges and obtains five subfraction Fr.2-5-1 – Fr.2-5-5; Component Fr.2-5-1 reclaims solvent, obtains compd A stin D(3 behind the evaporate to dryness); The Fr.3 component is that the eluent separation obtains two subfraction Fr.3-1 – Fr.3-2 through silica gel column chromatography with 1:1-1:2 sherwood oil/acetone; Wherein the Fr.3-2 component is that the eluent separation obtains two component Fr.3-2-1 and Fr.3-2-2 again through silica gel column chromatography with 1:3-1:5 chloroform/ethyl acetate; Fr.3-2-2 is through Sephadex LH-20 chromatography, and the chloroform/methanol of 1:1 is an eluent, obtains the part of enrichment cyclic peptide.This part obtains compd A stin C(1 through recrystallization); The Fr.4 component is through silica gel column chromatography, and 1:1-1:2 sherwood oil/acetone carries out gradient elution, merges similar cyclic peptide point part, obtains two subfraction Fr.4-1 – Fr.4-2; Subfraction Fr.4-2 is again through silica gel column chromatography, 2:1, and 1:1,1:2 sherwood oil/acetone carries out gradient elution, merges to obtain three subfraction Fr.4-2-1 – Fr.4-2-3; Wherein Fr.4-2-2 carries out the cyclic peptide enrichment through Sephadex LH-20, and the 1:1 chloroform/methanol is an eluent, again through RP-18 column chromatography enrichment cyclic peptide, 30:70-80:20 methanol gradient elution; Behind Sephadex LH-20 and RP-18 enrichment cyclic peptide, component Fr.4-2-2 is again through silica gel column chromatography, 20:1, and 10:1, the 5:1 chloroform/methanol is that eluent carries out gradient elution, obtains four subfraction Fr.4-2-2-1 – Fr.4-2-2-4; Wherein Fr.4-2-2-2 is again through silica gel column chromatography 20:1,10:1, and the 5:1 chloroform/methanol is that eluent carries out gradient elution, obtains five subfraction Fr.4-2-2-2-1 – Fr.4-2-2-2-5; Wherein directly through Sephadex LH-20 purification enrichment cyclic peptide part, the 1:1 chloroform/methanol is an eluent to Fr.4-2-2-2-4, merges the cyclic peptide part, reclaims solvent, and evaporate to dryness obtains compd A stin H(4); Component Fr.4-2-2-4 is through Sephadex LH-20 purification enrichment cyclic peptide part, and the 1:1 chloroform/methanol is an eluent, obtains two component Fr.4-2-2-4-1 – Fr.4-2-2-4-2; Component Fr.4-2-2-4-1 reclaims solvent, obtains compound Tataricin A(5 behind the evaporate to dryness); The Fr.5 component is through silica gel column chromatography, 1:3-1:9 chloroform/ethyl acetate gradient elution, and with the enrichment of cyclic peptide part, this part is through Sephadex LH-20, and the 1:1 chloroform/methanol is an eluent, obtains two component Fr.5-1 – Fr.5-2; Wherein component Fr.5-2 is through silica gel column chromatography, and the 15:1-3:1 chloroform/methanol is carried out gradient elution, obtains two component Fr.5-2-1 – Fr.5-2-2; Component Fr.5-2-1 reclaims solvent, obtains compd A stin B(2 behind the evaporate to dryness).
R
1, R
2=aryl or alkyl
R
3, R
4, R
5=hydroxyl or halogen atom
X, Y=unsaturated link(age) or saturated bond
With the salt of allowing on above-mentioned composite family type cyclic peptide of the present invention or its pharmacology is the immunosuppressor of effective constituent.
Be used for the immunosuppressive drug compositions, wherein contain the salt and the pharmaceutically acceptable carrier of allowing on the of the present invention above-mentioned composite family type cyclic peptide for the treatment of significant quantity or its pharmacology.
The present invention also provides the composite family type cyclic peptide compounds Astin that contains shown in the said structure formula for the treatment of significant quantity C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) or its pharmacology on the salt of allowing and the pharmaceutical composition of pharmaceutically acceptable carrier.
The present invention provides the application of the salt of allowing on composite family type cyclic peptide of the present invention or its pharmacology in the preparation immunosuppressor simultaneously.
With compd A stin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) or its pharmacology on the salt of allowing be the immunosuppressor of effective constituent.
Compd A stin C(1), the application in preparation immunosuppressor Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))).
Compd A stin C(1) is used to prepare immunosuppressor.
The present invention carries out the cyclic peptide chemical constitution study of system to composite family aster aster, utilizes multiple separation and purification means, comprises positive reversed-phase silica gel column chromatography, Sephadex LH-20 gel chromatography, and recrystallizations etc. have therefrom obtained a series of composite family type cyclic peptide.Afterwards, to main cyclic peptide composition Astin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) on Con A inductive activating T cell proliferation experiment, detect its restraining effect, and on the mouse immune colitis experimental model that trinitro-benzene-sulfonic acid causes, detect its therapeutic action.Discovery Astin C(1), Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))) all show certain immunosuppressive activity in vivo and in vitro, immunosuppressor for the novel plant source can be used for preparing immunosuppressive drug.
The salt of allowing on described composite family type cyclic peptide of the present invention or its pharmacology, for example can enumerate and mineral acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, Hydrogen bromide, perhaps organic acid such as toxilic acid, fumaric acid, tartrate, lactic acid, citric acid, acetate, methylsulfonic acid, right-benzene methanesulfonic acid, hexanodioic acid, palmitinic acid, Weibull, lithium, basic metal such as sodium, potassium, alkaline-earth metal such as calcium, magnesium, the salt that basic aminoacidss such as Methionin become.
The pharmaceutical composition of treatment immunity system relative disease of the present invention, by the pharmaceutical dosage form of composite family type cyclic peptide compounds and pharmaceutically acceptable preparing carriers comprise tablet, capsule, oral liquid, injection, injection is freeze-dried or powder injection etc.Since composite family type cyclic peptide can be from aster and congener extraction separation, and tablet, capsule, oral liquid, injection, injection is freeze-dried or the preparation of pharmaceutical dosage form such as powder injection also is the conventional knowledge of this area.Therefore.Various pharmaceutical dosage forms by composite family type cyclic peptide compounds and respective carrier preparation also can be realized by those skilled in the art.
Above described pharmaceutically acceptable carrier is meant the pharmaceutical carrier of pharmaceutical field routine, for example: thinner, vehicle such as water etc., weighting agent such as starch, sucrose etc.; Tamanori such as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent such as glycerine; Disintegrating agent such as agar, lime carbonate and sodium bicarbonate; Absorption enhancer such as quaternary ammonium compound; Tensio-active agent such as cetyl alcohol; Absorption carrier such as kaolin and soap clay; Lubricant such as talcum powder, calcium stearate and Magnesium Stearate and polyoxyethylene glycol etc.Can also in composition, add other assistant agent such as flavouring agent, sweeting agent etc. in addition.
The compounds of this invention can with the form of composition by oral, snuffing is gone into, the mode of rectum or administered parenterally is applied to the patient who needs this treatment.Be used for when oral, can be made into conventional solid preparation such as tablet, pulvis, granula, capsule etc., make liquid preparation such as water or oil-suspending agent or other liquid preparations such as syrup, elixir etc.; When being used for administered parenterally, can be made into solution, water or the oiliness suspension agent etc. of injection.The various formulations of pharmaceutical composition of the present invention can be according to the conventional production method preparation of pharmaceutical field.Activeconstituents is mixed with one or more carriers, be made into required formulation then.
It is 0.1%～99.5% activeconstituents that pharmaceutical composition of the present invention preferably contains weight ratio, most preferably contains weight ratio and be 0.5%～95% activeconstituents.
The amount of application of The compounds of this invention can be according to variations such as the type of route of administration, patient's age, body weight, the disease of being treated and severity, and its per daily dose can be 0.01～10 mg/kg body weight, preferred 0.1～5 mg/kg body weight.Can use by one or many.
The excellent benefit of composite family type cyclic peptide extracting method of the present invention is, at first the extraction separation thinking has novelty, successfully utilize the chromatography technology that other small molecules are separated with composite family type cyclic peptide, obtain the comparatively cyclic peptide of purifying, utilize technical points such as recrystallization again from the cyclic peptide that obtains purifying.Put it briefly, because of the main chemical compositions of aster is terpene and glycoside thereof, in the cyclic peptide sepn process, have a large amount of terpenoids to disturb, especially triterpenoid saponin is usually mixed in together not easily separated with cyclic peptide.So at first utilize the molecular sieve principle of Sephadex LH-20, can separate the ter penoids that a part and cyclic peptide molecular weight differ greatly, utilize the adsorption chromatography principle of normal phase column chromatographic column again, the success separation obtains containing the less cyclic peptide part of pigment, comprise that in conjunction with various chromatographic materials RP-18 and multiple separation means comprise recrystallization etc. again, separation and purification is to the pure product of cyclic peptide.Secondly, only utilize the chromatographic material of laboratory or industrial routine, comprise positive reverse phase silica gel, Sephadex LH-20 etc., needn't use special chromatographic material such as aminopropyl key and silica gel, also avoid using chromatographic materials such as stronger activated carbon of adsorptivity and aluminum oxide; At last, in the sepn process of cyclic peptide must in conjunction with the TLC detection method of cyclic peptide (referring to Zhou Jun, Tan Ninghua, Science Bulletin, 2000,45(10), 1047-1051).In a word, extraction and separation method controllability of the present invention and favorable reproducibility, sample loss is few, and cost is lower, and is easy to operate, separablely obtains micro-cyclic peptide, and solvent can be recycled repeatedly, also is applicable to industrial production.
Description of drawings:
Fig. 1 is preparation method's schema of composite family type cyclic peptide compounds of the present invention;
Fig. 2 a is the effect of composite family type cyclic peptide compounds Astin C of the present invention to normal T cell proliferation; Fig. 2 b is the effect of composite family type cyclic peptide compounds Astin C of the present invention to activating T cell propagation;
The therapeutic action of Fig. 3 a, Fig. 3 b, Fig. 3 c mouse immune colitis experimental model that to be composite family type cyclic peptide compounds Astin C of the present invention cause trinitro-benzene-sulfonic acid.
Embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.Essence according to the present invention all belongs to scope of the present invention to the improvement that the present invention carries out.
Embodiment 1:
Composite family type cyclic peptide compounds Astin C(1), Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))) preparation and Tataricin A(5) structure identify:
Get aster (
Aster tataricus. L) dry root and rhizome 50 Kg, pulverize the back with the first cold soaking of 90% industrial methanol 5 hours, post-heating refluxing extraction (temperature=65 ℃), extract altogether three times, 3 hours for the first time, 2 hours for the second time, 2 hours for the third time, united extraction liquid, underpressure distillation gets methyl alcohol medicinal extract totally 23 Kg after concentrating and removing organic solvent, and this crude extract is allocated in the water, fully extracts with ethyl acetate, propyl carbinol successively, equal-volume respectively extracts three times, reclaims solvent and obtains ethyl acetate part (2Kg), propyl carbinol part (5L) and water section; Ethyl acetate is partly used dissolve with methanol, and 2.5 Kg 100-200 order silica gel mixed samples are after sample drying is mixed by institute, sieve respectively with sieve aperture 40 orders and 80 purpose sieves, sieve the back sample on silicagel column (the 200-300 order 10Kg), is used 100:0,9:1,8:2,7:3, the chloroform/methanol gradient elution of 0:100, thin-layer chromatography detects with the specific coloration method of cyclic peptide-triketohydrindene hydrate in-situ chemical reaction method.According to the result of cyclic peptide colour developing, merge the part that 9:1 and 8:1 contain cyclic peptide, altogether 1063g.Each following step all must be carried out separation and purification in conjunction with cyclic peptide TLC detection method.Medicinal extract after the merging is once more through silica gel column chromatography, 950g 100-200 order silica gel mixed sample, with (20:1,10:1,85:15,8:2,0:1) chloroform/methanol gradient elution is merged into eight components (Fr.1-Fr.8) according to the difference of cyclic peptide point; Fr.2(75 g) component is through silica gel column chromatography, and the sherwood oil of 2:1-1:2/acetone gradient elution merges and obtains six subfraction Fr.2-1 – Fr.2-6; Fr.2-5(4 g) again through silica gel column layer, 20:1,15:1,10:1, the chloroform/methanol gradient elution of 5:1, merging obtains five subfraction Fr.2-5-1 – Fr.2-5-5; Component Fr.2-5-1(1.5 g) reclaim solvent, obtain compd A stin D(3 behind the evaporate to dryness) (1 g).
Component Fr.3(55 g), be that the eluent separation obtains two subfraction Fr.3-1 – Fr.3-2 with 1:1-1:2 sherwood oil/acetone through silica gel column chromatography; Fr.3-2(40 g wherein) component is again through silica gel column chromatography, is that eluent separates and obtains two component Fr.3-2-1 and Fr.3-2-2 with 1:3-1:5 chloroform/ethyl acetate; Fr.3-2-2(24 g) through Sephadex LH-20 chromatography, the chloroform/methanol of 1:1 is an eluent, obtains the part of enrichment cyclic peptide.This part obtains compd A stin C(1 through recrystallizing methanol) (8 g).
Component Fr.4(85 g) through silica gel column chromatography, 1:1-1:2 sherwood oil/acetone carries out gradient elution, merges similar cyclic peptide point part, obtains two subfraction Fr.4-1 – Fr.4-2; Subfraction Fr.4-2(60 g) again through silica gel column chromatography, 2:1,1:1,1:2 sherwood oil/acetone carries out gradient elution, merges to obtain three subfraction Fr.4-2-1 – Fr.4-2-3; Fr.4-2-2(14 g wherein) carry out the cyclic peptide enrichment through Sephadex LH-20, the 1:1 chloroform/methanol is an eluent, again through RP-18 column chromatography enrichment cyclic peptide, 30:70-80:20 methanol gradient elution; Behind Sephadex LH-20 and RP-18 enrichment cyclic peptide, component Fr.4-2-2 is again through silica gel column chromatography, 20:1, and 10:1, the 5:1 chloroform/methanol is that eluent carries out gradient elution, obtains four subfraction Fr.4-2-2-1 – Fr.4-2-2-4; Fr.4-2-2-2(4 g wherein) again through silica gel column chromatography 20:1,10:1, the 5:1 chloroform/methanol is that eluent carries out gradient elution, obtains five subfraction Fr.4-2-2-2-1 – Fr.4-2-2-2-5; Fr.4-2-2-2-4(210mg wherein) directly through Sephadex LH-20 purification enrichment cyclic peptide part, the 1:1 chloroform/methanol is an eluent, merges the cyclic peptide part, reclaims solvent, and evaporate to dryness obtains compd A stin H(4) (120 mg); Component Fr.4-2-2-4(1.5 g) through Sephadex LH-20 purification enrichment cyclic peptide part, the 1:1 chloroform/methanol is an eluent, obtains two component Fr.4-2-2-4-1 – Fr.4-2-2-4-2; Component Fr.4-2-2-4-1(55 mg) reclaim solvent, obtain compound Tataricin A(5 behind the evaporate to dryness) (40 mg);
Fr.5(146 g) component is through silica gel column chromatography, 1:3-1:9 chloroform/ethyl acetate gradient elution, and with the enrichment of cyclic peptide part, this part is through Sephadex LH-20, and the 1:1 chloroform/methanol is an eluent, obtains two component Fr.5-1 – Fr.5-2; Component Fr.5-2(50 g wherein) through silica gel column chromatography, the 15:1-3:1 chloroform/methanol is carried out gradient elution, obtains two component Fr.5-2-1 – Fr.5-2-2; Component Fr.5-2-1(17 g) reclaim solvent, obtain compd A stin B(2 behind the evaporate to dryness) (15 g).
R
1, R
2=aryl or alkyl
R
3, R
4, R
5=hydroxyl or halogen atom
X, Y=unsaturated link(age) or saturated bond
Tataricin A(5) structure appraising datum is:
Tataricin A(5): clear crystal;
-23.2 (
c0.19, C
5H
5N); UV (MeOH)
λ Max(log e) 204 (4.26), 268 (4.20) nm; IR (KBr)
n Max3423,1660,1533,1409,1305,1149,1075,752,578 cm
-1;
1H NMR (DMSO-
d 6 , 600 MHz) and and
13C NMR (DMSO-
d 6 , 150 MHz), data see Table 1; Positive ESIMS
M/z514 (30) [M+H]
+; Positive HRESIMS
M/z[514.2295 M+H]
+(cacld for C
25H
32N
5O
7,514.2301).
Embodiment 2:
Composite family type cyclic peptide compounds Astin C(1 of the present invention), Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))) show that on Con A inductive activating T cell proliferation experiment inhibition is active.Experimental principle, method and result are as follows:
Experimental principle: the T lymphocyte of normal body is subjected to specific antigens or mitotic division primary stimuli in the vitro culture process, a series of variations can take place for the metabolism of cell and form.Mainly show as the change of electric charge, cell volume increases, and metabolism is vigorous, and intracellular protein and nucleic acid are synthetic to increase (and can divide), and cellular form can be converted into lymphoblast, is the lymphocyte transformation phenomenon.For this reason, utilize various stimulants to excite lymphocyte, can measure the lymphocytic answering of T (height of lymphocyte transformation rate can reflect the body cell immune level) according to its transforming degree external.This experimental applications Con A stimulates the T lymphocyte, measures the cell survival amount with mtt assay then.
Experimental technique: get 5 * 10
5The C57BL/6 mouse lymph nodal cell of cells/well places 96 well culture plates, and it is the Con A solution of 5 μ g/ml that medicine group and Control group add final concentration, puts 37 ℃, 5% CO
2In the incubator, cultivate 24 h.Adopt MTT to survey cell survival rate behind 24 h.The record result is an X-coordinate with concentration, and the T cell survival rate is the ordinate zou curve plotting, the IC of computerized compound
50Value.
Experimental result:
Astin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) normal T cell proliferation is not all had remarkable restraining effect, compd A stin C demonstrates certain restraining effect to activating T cell propagation, concrete IC
50See Table 1.
Experimental result shows: aster type cyclic peptide Astin C demonstrates certain restraining effect, IC to activating T cell propagation
50=12 μ M.When 20 μ M, inhibiting rate is near S-Neoral (CsA).Because the low cytotoxicity of aster cyclic peptide Astin C, thereby possesses the prospect that becomes new cyclic peptide immunosuppressor.
Embodiment 3:
Composite family type cyclic peptide compounds Astin C(1 of the present invention), the therapeutic action of the mouse immune colitis that trinitro-benzene-sulfonic acid (TNBS) is caused Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))).Experimental principle, method and result are as follows:
Experimental principle: inflammatory bowel mainly comprises ulcerative colitis and Crohn's disease, is a kind of chronic nonspecific enteritis disease of outbreak repeatedly, and the cause of disease that it is definite and pathogenesis are not clear so far, also lack safe and effective medicine in the treatment.Trinitro-benzene-sulfonic acid inductive mouse colitis is short because of its cycle, and method is easy, is the immunologic mechanism of the human inflammatory bowel of research and the ideal model of research and development anti-inflammatory new drug.
Experimental technique: got for 6 ~ 8 ages in week, the C57BL/6 female mice of 18 ~ 22 g carefully inserts colon with 3.5 F flexible pipes via anus, about 4 cm of depth of penetration.With 1 mL disposable sterilized injector TNBS is injected colon through flexible pipe, every injected in mice TNBS dosage is 100 mg/kg, and solvent is 40% ethanol PBS solution, after the injection, mouse was stood upside down 30 seconds, spill to prevent TNBS, and make it to be distributed in whole colon, comprise caecum and appendix.The blank group is injected 100 μ L solvents (40% ethanol PBS solution) with identical method.From modeling same day, every day oral administration once, continue ten days, finish until experiment.From modeling same day, weighing every day mouse body weight, diarrhoea situation and the death condition of every group of mouse are observed and write down to record the compare increase or the reduction of modeling body weight on same day value simultaneously.After experiment finishes, put to death mouse and get colon, write down each treated animal colon length and add up and observe its tissue pathologies change, get animal serum, measure the level of inflammatory cytokine in the serum.
Experimental result: Astin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) can alleviate alleviating of TNBS inductive colitis mice body weight; Improve TNBS inductive colitis mice survival rate; Alleviate TNBS inductive colitis mice diarrhoea; The oedema that alleviates TNBS colitis mice colon shortens; Reduce the lesion degree of TNBS colitis mice colon; The level that suppresses Th1 cytokine TNF-α in the TNBS colitis mice serum.
Experimental result shows, Astin C(1), Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))) mouse colitis tool that trinitro-benzene-sulfonic acid is caused has some improvement, and proves Astin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) can become a kind of neotype immunosuppressant of treatment inflammatory bowel.
Embodiment 4:
Embodiment 5:
Embodiment 6:
Embodiment 7:
Embodiment 8:
Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))) or salt 10 mg of embodiment 4-7 gained tablet: with embodiment 1 gained compd A stin C(1),, lactose 180 mg, starch 55 mg, Magnesium Stearate 5 mg, lactose and starch are mixed, and water is evenly moistening, the mixture after moistening is sieved and drying, after sieve, add Magnesium Stearate, then with the mixture compressing tablet, every heavy 250 mg, compounds content is 10 mg.
Embodiment 9:
Tataricin A(5 Astin H(4 Astin D(3 Astin B(2)))) or salt 2 mg of embodiment 4-7 gained ampulla: with embodiment 1 gained compd A stin C(1),, sodium-chlor 10 mg, be dissolved in the proper amount of water for injection, filter gained solution, in the ampoule of under aseptic condition, packing into.
Embodiment 10:
Injection is freeze-dried: embodiment 1 gained compd A stin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) or salt 10 mg of embodiment 4-7 gained, sodium bicarbonate 2 mg, N.F,USP MANNITOL 252 mg.Preparation method: with sodium bicarbonate, N.F,USP MANNITOL, add the dissolving of injection water, add activated carbon adsorption 30 min depyrogenations, remove by filter activated carbon, add compound or its salt in filtrate, supersound process makes dissolving, regulating PH with 1 N hydrochloric acid is 5.0-7.0, millipore filtration filters, and adds the injection water, packing, lyophilize, top plug rolls lid, promptly.
Embodiment 11:
Capsule: embodiment 1 gained compd A stin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) or the salt 10mg of embodiment 4-7 gained, lactose 187 mg, Magnesium Stearate 3mg; The preparation method: compound or its salt and solubility promoter is mixed, sieve, uniform mixing, the mixture that the obtains hard gelatin capsule of packing into, each capsule weighs 200 mg, and active component content is 10 mg.
Claims (9)
1. composite family type cyclic peptide compounds is for skeleton contains
β-phenylalanine, and, contain a L-with the equal pentapeptide compound of monocycle that normal amino acid peptide bond directly links to each other and forms
β-phenylalanine, a L-Serine, a L-proline derivative and two other amino acid derivative.
2. cyclic peptide as claimed in claim 1 is for having the compd A stin C(1 of following structural formula), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5),
R
1, R
2=aryl or alkyl
R
3, R
4, R
5=hydroxyl or halogen atom
X, Y=unsaturated link(age) or saturated bond
3. the method for preparing the described composite family type of claim 1 cyclic peptide compounds is got composite family aster rhizome, after drying, the pulverizing, fully extracts with methanol eddy; After methyl alcohol medicinal extract added the water suspendible, fully extract with ethyl acetate and propyl carbinol successively; Ethyl acetate part and propyl carbinol part are used silica gel, the various chromatography column separation and purification of Sephadex LH-20, RP-18 repeatedly, and the TLC detection method in conjunction with cyclic peptide gets composite family type cyclic peptide compounds again.
4. be prepared as follows the type of composite family shown in structural formula cyclic peptide compounds Astin C(1), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) method, get the aster rhizome, after drying, the powder essence, extract 3 times with methanol eddy, time is each 1-3 hour, and extracting solution gets methyl alcohol medicinal extract through concentrating under reduced pressure; After methyl alcohol medicinal extract added the water suspendible, fully extract with ethyl acetate and propyl carbinol successively, equal-volume respectively extracts three times, reclaims solvent and obtains ethyl acetate part, propyl carbinol part and water section; Ethyl acetate part through silica gel column chromatography, use 100:0,9:1,8:2,7:3, the chloroform/methanol gradient elution of 0:100, thin-layer chromatography is in conjunction with cyclic peptide TLC detection method merging 9:1 and the 8:1 part that contains cyclic peptide wherein; Each following step all must be carried out separation and purification in conjunction with cyclic peptide TLC detection method; Medicinal extract after the merging is used 20:1 once more through silica gel column chromatography, 10:1, and 85:15,8:2, the chloroform/methanol gradient elution of 0:1 is merged into eight component Fr.1-Fr.8 according to the difference of cyclic peptide point; The Fr.2 component is through silica gel column chromatography, and the sherwood oil of 2:1-1:2/acetone gradient elution merges and obtains six subfraction Fr.2-1 – Fr.2-6; Fr.2-5 is again through silica gel column chromatography, 20:1, and 15:1,10:1, the chloroform/methanol gradient elution of 15:1 merges and obtains five subfraction Fr.2-5-1 – Fr.2-5-5; Component Fr.2-5-1 reclaims solvent, obtains compd A stin D(3 behind the evaporate to dryness); The Fr.3 component is that the eluent separation obtains two subfraction Fr.3-1 – Fr.3-2 through silica gel column chromatography with 1:1-1:2 sherwood oil/acetone; Wherein the Fr.3-2 component is that the eluent separation obtains two component Fr.3-2-1 and Fr.3-2-2 again through silica gel column chromatography with 1:3-1:5 chloroform/ethyl acetate; Fr.3-2-2 is through Sephadex LH-20 chromatography, and the chloroform/methanol of 1:1 is an eluent, obtains the part of enrichment cyclic peptide; This part obtains compd A stin C(1 through recrystallization); The Fr.4 component is through silica gel column chromatography, and 1:1-1:2 sherwood oil/acetone carries out gradient elution, merges similar cyclic peptide point part, obtains two subfraction Fr.4-1 – Fr.4-2; Subfraction Fr.4-2 is again through silica gel column chromatography, 2:1, and 1:1,1:2 sherwood oil/acetone carries out gradient elution, merges to obtain three subfraction Fr.4-2-1 – Fr.4-2-3; Wherein Fr.4-2-2 carries out the cyclic peptide enrichment through Sephadex LH-20, and the 1:1 chloroform/methanol is an eluent, again through RP-18 column chromatography enrichment cyclic peptide, 30:70-80:20 methanol gradient elution; Behind Sephadex LH-20 and RP-18 enrichment cyclic peptide, component Fr.4-2-2 is again through silica gel column chromatography, 20:1, and 10:1, the 5:1 chloroform/methanol is that eluent carries out gradient elution, obtains four subfraction Fr.4-2-2-1 – Fr.4-2-2-4; Wherein Fr.4-2-2-2 is again through silica gel column chromatography 20:1,10:1, and the 5:1 chloroform/methanol is that eluent carries out gradient elution, obtains five subfraction Fr.4-2-2-2-1 – Fr.4-2-2-2-5; Wherein directly through Sephadex LH-20 purification enrichment cyclic peptide part, the 1:1 chloroform/methanol is an eluent to Fr.4-2-2-2-4, merges the cyclic peptide part, reclaims solvent, and evaporate to dryness obtains compd A stin H(4); Component Fr.4-2-2-4 is through Sephadex LH-20 purification enrichment cyclic peptide part, and the 1:1 chloroform/methanol is an eluent, obtains two component Fr.4-2-2-4-1 – Fr.4-2-2-4-2; Component Fr.4-2-2-4-1 reclaims solvent, obtains compound Tataricin A(5 behind the evaporate to dryness); The Fr.5 component is through silica gel column chromatography, 1:3-1:9 chloroform/ethyl acetate gradient elution, and with the enrichment of cyclic peptide part, this part is through Sephadex LH-20, and the 1:1 chloroform/methanol is an eluent, obtains two component Fr.5-1 – Fr.5-2; Wherein component Fr.5-2 is through silica gel column chromatography, and the 15:1-3:1 chloroform/methanol is carried out gradient elution, obtains two component Fr.5-2-1 – Fr.5-2-2; Component Fr.5-2-1 reclaims solvent, obtains compd A stin B(2 behind the evaporate to dryness),
R
1, R
2=aryl or alkyl
R
3, R
4, R
5=hydroxyl or halogen atom
X, Y=unsaturated link(age) or saturated bond
5. pharmaceutical composition, wherein contain the composite family type cyclic peptide compounds Astin C(1 shown in the following structural formula for the treatment of significant quantity), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) or its pharmacology on the salt and the pharmaceutically acceptable carrier of allowing
R
1, R
2=aryl or alkyl
R
3, R
4, R
5=hydroxyl or halogen atom
X, Y=unsaturated link(age) or saturated bond
。
6. be the immunosuppressor of effective constituent with the salt of allowing on the composite family type cyclic peptide in the claim 1 or its pharmacology.
7. with the compd A stin C(1 in the claim 2), Astin B(2), Astin D(3), Astin H(4), Tataricin A(5) or its pharmacology on the salt of allowing be the immunosuppressor of effective constituent.
8. the application of the salt of allowing on claim 1 described composite family type cyclic peptide or its pharmacology in the preparation immunosuppressor.
9. the application in the preparation immunosuppressor Tataricin A(5 Astin H(4 Astin D(3 Astin B(2 compd A stin C(1 in the claim 2))))).
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| CN110357864A (en) * | 2019-05-22 | 2019-10-22 | 中国科学院昆明植物研究所 | A kind of pyrroles's sesquiterpenoids and its preparation method and application |
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| CN107335049A (en) * | 2017-08-18 | 2017-11-10 | 中国药科大学 | Application of the composite family type cyclic peptide compounds as cGAS STING signal pathway inhibitors |
| WO2019033710A1 (en) * | 2017-08-18 | 2019-02-21 | 中国药科大学 | Use of compositae-type cyclic peptide compound as cgas-sting signalling pathway inhibitor |
| CN107335049B (en) * | 2017-08-18 | 2019-10-18 | 中国药科大学 | Application of the composite family type cyclic peptide compounds as cGAS-STING signal pathway inhibitor |
| JP2020531482A (en) * | 2017-08-18 | 2020-11-05 | 中国▲薬▼科大学Ｃｈｉｎａ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｕｎｉｖｅｒｓｉｔｙ | Application of Asteraceae cyclic peptide compounds as cGAS-STING signaling pathway inhibitors |
| CN110054665A (en) * | 2019-04-29 | 2019-07-26 | 湖南新汇制药股份有限公司 | A kind of five titanium compound of ring, preparation method and its preparing the application in immune suppressant drug |
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| CN110357864A (en) * | 2019-05-22 | 2019-10-22 | 中国科学院昆明植物研究所 | A kind of pyrroles's sesquiterpenoids and its preparation method and application |
| CN110357864B (en) * | 2019-05-22 | 2022-06-14 | 中国科学院昆明植物研究所 | Pyrrole sesquiterpenoid compound and preparation method and application thereof |
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