CN102816212B

CN102816212B - Rubiaceae-type cyclopeptide, pharmaceutical composition and application thereof

Info

Publication number: CN102816212B
Application number: CN201210306178XA
Authority: CN
Inventors: 谭宁华; 范君婷; 李艳; 苏佳; 李佳; 周宇波; 彭延敏; 彭涛; 朱钦昌; 曾广智
Original assignee: Kunming Institute of Botany of CAS; Shanghai Institute of Materia Medica of CAS; Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Kunming Institute of Botany of CAS; Shanghai Institute of Materia Medica of CAS; Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2010-07-21
Filing date: 2010-07-21
Publication date: 2013-11-13
Anticipated expiration: 2030-07-21
Also published as: CN102816212A

Abstract

The present invention provides Rubiaceae type cyclic peptide compounds rubiyunnanin F(8), using it as the pharmaceutical composition of active constituent, preparation method and application. The preparation method of this compound is provided and is preparing the application in anti-herpes simplex virus I type (HSV-1) drug.

Description

Rubiaceae type cyclic peptide and pharmaceutical composition thereof and application

The application be patent application " Rubiaceae type cyclic peptide, the pharmaceutical composition take it as activeconstituents, its preparation method and application " (application number: 201010232742.9, the applying date: dividing an application on July 21st, 2010).

Technical field:

The invention belongs to technical field of pharmaceuticals, particularly, relate to a class Rubiaceae type cyclic peptide, as NF-κ B signal pathway inhibitor, pharmaceutical composition take it as effective constituent, its preparation method and the application in preparing anti-herpes simplex virus I type (HSV-1) medicine thereof.

Background technology:

NF-κ B signal path wide participation various biological effect, comprise immune response, inflammatory reaction, virus infection, Growth of Cells, apoptosis and tumor development etc., is one of most important signal path in the higher organism cell.Nuclear Factor kappa B is the crucial transcription factor in immunne response, inflammatory reaction and body development process, and its location and vigor in cell is subject to strict regulation and control.The NF-κ B of activation participates in the genetic expression of the multiple factor of regulation and control, as causes inflammatory reaction factor NO, TNF-α, IL-2, IL-6, IL-8, chemokine MCP-1, RANTES, cell adhesion molecule ICAM-1, VCAM-1 etc., if these regulate abnormal, will cause the pathological phenomenons such as microbial infection, autoimmune disease, heteroplasia and tumour.NF-κ B family protein is ubiquity in all kinds cell in vivo, mainly contains 5 members in mammalian cell, is respectively RelA(P65), RelB, c-Rel, NF-κ B1(p50/p105) and NF-κ B2(p52/p100).Each member can form homotype or heterodimer and exist, and modal is the p65/p50 dimer.NF-κ B signal path mainly comprises a classical signals path and two non-classical signal paths.

At present existing many NF-κ B signal pathway inhibitors are found, and by sources mainly are divided into natural product, synthetic compound, SiRNA, polypeptide and albumen (virus, bacterium, fungi) etc.Active position according to inhibitor in path and mechanism of action difference can be divided into act on IKK complex body upstream, directly for four classes such as between IKK complex body, IKK and nucleus, in nucleus.In addition, NF-κ B pathway inhibitor is existing by the successful case of FDA approval listing as clinical medicine, the white arsenic (ATO) that for example is used for the treatment of tumour, Thalidomide, proteasome inhibitor PS341(bortezomib) and TNF-alpha monoclonal antibodies Remicade and the Humira of the inflammation related diseases such as treatment rheumatoid disease sacroiliitis.But the clinical application report of the micromolecular inhibitor of other binding modes, novel structure is also less, and mechanism of action and the regulatory mechanism of this path in the relative disease genesis remains more deeply to be illustrated simultaneously.So find that the NF-κ B signal path micromolecular inhibitor of novel structure is used for the treatment of this path relative disease, or find that specific tool molecule or probe molecule go to illustrate the Role in Plant Signal Transduction mechanism of this path, have very important academic significance and good application prospect.

Hsv (herpes simplex virus, HSV) belongs to the after birth DNA virus of Alphaherpesviridae in herpetoviridae (herpesviridae).Clinically, HSV has two serotypes, is respectively HSV-1 and HSV-2.HSV-1 mainly causes mucous membrane and the nervous system infection that people's waist is above, and associated diseases has keratitis, herpes simplex encephalitis, meningitis, herpes labialis and gingivostomatitis etc.HSV-2 mainly causes the following genital infection of waist, is the main pathogens of genital herpes (GH).HSV congenital infection and infection of newborn, can cause fetus congenital malformation, miscarriage, premature labor and newborn infant's herpetic disease.HSV is to adult's infection rate up to 60%～90%, and its most significant biological characteristics is to hole up in the body after primary infection, and sustainable existence.The stimulation of the factors such as high heat, immunosuppression, operation can activate the HSV that hides, and causes symptomatic outbreak, or the viral spread of silent.HSV can also cause cell carcinogenesis, and it infects separately, or with the coinfection of human papillomavirus (HPV) be the one of the main reasons that cervical cancer occurs.

Successful HSV vaccine listing is not yet arranged at present.Be used for the treatment of the medicine of HSV infection take nucleoside derivate as main, thereby be mainly by with the normal substrate competition inhibition of viral dna polymerase viral DNA, synthesizing and reach antiviral purpose, as Acyclovir (ACV), Famciclovir (FCV), Lip river of beautifully adorned former times big (PCV) and Ganciclovir (GCV) etc.These medicines have good curative effect to treatment HSV, but because mechanism of action is single, along with the frequency of using and the increase of dosage, the problem of resistance and toxic side effect becomes increasingly conspicuous.Medicinal plant is the important sources of developing new drug,, from finding new anti-herpesvirus medicament natural product widely, especially has the medicine of different mechanism of action, has extremely important realistic meaning.

In prior art, there are no the report of Rubiaceae type cyclic peptide as NF-κ B signal pathway inhibitor, in addition, Rubiaceae type cyclic peptide also has no report to the activity inhibition of herpes simplex virus I-type;

The separation and purification of Rubiaceae type cyclic peptide in madder wort be difficulty comparatively, most important reason is to be rich in anthraquinone pigments in Rubia plants, and pigment polarity scope is wide, has from the fat-soluble pigment to the water colo(u)r, is difficult to make this type of pigment and cyclic peptide composition to separate.In prior art, the separating ring peptide constituents has been published following method from Rubia plants: (1) seventies, cold fourth of cold fourth of Rubiaceae genus flower, stem, leaf extract with methyl alcohol and acetonitrile successively, separate out precipitation with methyl alcohol and isopropyl ether low temperature again, separate and obtain cyclic peptide bouvardin and deoxybouvardin(RA-V with preparation TLC afterwards).Referring to: Jolad, S.D.et al, Bouvardin and deoxybouvardin, antitumor cyclic hexapeptides from Bouvardia ternifolia (Rubiaceae), J.Am.Chem.Soc., 1977,99 (24), 8040-8044.Its shortcoming is that acetonitrile solvent is more expensive as extracting the solvent price, is unsuitable for industrial production; Low-temperature sludge technological controllability and circulation ratio are bad; Only with preparation TLC technology, be difficult to obtain micro-cyclic peptide.(2) nineties, the rhizome of Rubiaceae Rubia Radix Rubiae Yunnanensis with methanol extraction after, methanol extract, through activated carbon, silica gel or alkylated silica gel column chromatography separating purification, obtains cyclic peptide RA-V, RA-XII and RY-II.Referring to: Zou Cheng etc., the anticancer Cyclohexapeptides glycoside of Radix Rubiae Yunnanensis, Yunnan plant research, 1993,15(4), 399-402.Its shortcoming is that activated carbon is strong to the adsorptivity of sample, easily loses sample, and is difficult to obtain micro-cyclic peptide.(3) in recent years, the root of Rubiaceae Rubia madder with methanol extraction after, methanol extract is used chloroform extraction after adding the water suspendible, chloroform part obtains the cyclic peptide enriching section with silica gel, aluminum oxide, aminopropyl key and silica gel chromatography column chromatography successively.This cyclic peptide enriching section recrystallizing methanol, then in conjunction with various chromatographic materials, comprise that HPLC carries out separation and purification to crystallization or mother liquor, obtain a series of cyclic peptide, comprise a large amount of and micro-cyclic peptide.Referring to: Lee, J.E.et al.Structures of cytotoxic bicyclic hexapeptides, RA-XIX ,-XX ,-XXI, and – XXII, from Rubia cordifolia L.Tetrahedron., 2008,64,4117-4125.Its shortcoming is that aluminum oxide is strong to the adsorptivity of sample, and aminopropyl key and silica gel material price are more expensive, laboratory and industrial being of little use, and recrystallizing methanol technological controllability and circulation ratio are bad.

Summary of the invention:

, for prior art above shortcomings part, the object of the present invention is to provide a class Rubiaceae 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, separablely obtains micro-cyclic peptide, and solvent can be recycled repeatedly, also is applicable to industrial production; The present invention also aims to provide the pharmaceutical composition take Rubiaceae type cyclic peptide compounds as effective constituent; This compounds is as novel NF-κ B signal pathway inhibitor, and the application in the medicine for preparing anti-herpes simplex virus I type.

, in order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:

Rubiaceae type cyclic peptide compounds, by 1 D-α-alanine, 1 L-α-alanine, the L-a-amino acid of the N-methyl of 3 replacements-L-α-tyrosine and 1 other kind forms through the peptide bond condensation, and 6 amino acid condensations become eighteen ring, and wherein the phenyl ring between the tyrosine at 2 ortho positions is connected to form a fourteen-ring through oxo bridge.

Cyclic peptide of the present invention, the compound R A-V(1 that preferably has following structural formula), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9), rubiyunnanin H(10) and RY-II(11).

The method for preparing the above-mentioned Rubiaceae type of the present invention cyclic peptide compounds, get the rhizome of Rubia plants, drying, pulverize after, fully extract with methanol eddy; After methanol extract is added the water suspendible, fully extract with ethyl acetate and propyl carbinol successively; Ethyl acetate part and propyl carbinol part are used silica gel, Sephadex LH-20 repeatedly, RP-18, and silica gel H, the various chromatography column separation and purification of HPLC, then in conjunction with the TLC detection method of cyclic peptide, namely obtain Rubiaceae type cyclic peptide.

Be prepared as follows the type of Rubiaceae shown in structural formula cyclic peptide compounds RA-V(1), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9) and rubiyunnanin H(10) and method RY-II(11), get the rhizome of Radix Rubiae Yunnanensis, after drying, pulverizing, extract 3 times with methanol eddy, time is each 1-4 hour, and extracting solution obtains methanol extract through concentrating under reduced pressure.After methanol extract is 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 100:0,95:5,9:1,8:2, the chloroform/methanol gradient elution of 0:100, thin-layer chromatography, in conjunction with cyclic peptide TLC detection method merging 95:5,9:1 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 merging, again through silica gel column chromatography,, with the chloroform/methanol gradient elution of 70:1-8:2, is merged into six component Fr.1-Fr.6 according to the difference of cyclic peptide point.The Fr.1 component, through silica gel column chromatography, is that the eluent separation obtains five subfraction Fr.1-1 – Fr.1-5 with 7:3-0:1 sherwood oil/acetone.Wherein Fr.1-4 is through Sephadex LH-20 chromatography, and the chloroform/methanol of 1:1 is eluent, and the part that contains cyclic peptide of institute's enrichment is again through silica gel H chromatography repeatedly, with the sherwood oil of 6:4/acetone wash-out, separates and obtains compound R A-V(1).The Fr.2 component is through silica gel column chromatography, and the chloroform/methanol gradient elution of 95:5-9:1, merge and obtain seven subfraction Fr.2-1 – Fr.2-7.Fr.2-4 partly uses anti-phase RP-18 silica gel mixed sample, and through the RP-18 column chromatography, the methanol/water gradient elution with 20:80-90:10, obtain six subfraction Fr.2-4-1 – Fr.2-4-6.Component Fr.2-4-2 is through Sephadex LH-20 chromatography, and the chloroform/methanol of 1:1 is eluent, and the cyclic peptide part of enrichment is again through the RP-18 column chromatography, and the methanol/water gradient elution of 30:70-50:50 obtains compound R A-I(2).Component Fr.2-4-5 is equally through Sephadex LH-20 chromatography, the chloroform/methanol of 1:1 is eluent, the cyclic peptide part of enrichment is through anti-phase RP-18 column chromatography, the methanol/water gradient elution of 20:80-40:60, contain cyclic peptide part again through silica gel H chromatography repeatedly, with the separation of 20:1-10:1 chloroform/methanol wash-out, obtain compound rubiyunnanin C(6).The Fr.3 component is through silica gel column chromatography, and the chloroform/methanol gradient elution of 70:1-9:1, merge and obtain five subfraction Fr.3-1 – Fr.3-5; The Fr.3-3 component is again through silica gel column chromatography, 1:1-0:1 sherwood oil/acetone gradient elution, contain the cyclic peptide part through Sephadex LH-20 enrichment again, the 1:1 chloroform/methanol is eluent, the cyclic peptide of institute's enrichment part is through silica gel H purifying repeatedly, the 20:1 chloroform/methanol is eluent, separates and obtains compound R A-XXIV(3).The Fr.4 component is through silica gel column chromatography, and the 20:1-8:2 ethyl acetate/methanol is carried out gradient elution, merges and obtains four subfraction Fr.4-1 – Fr.4-4.Subfraction Fr.4-3 directly carries out the cyclic peptide enrichment through Sephadex LH-20, and the 1:1 chloroform/methanol is eluent.Through silica gel column chromatography, 95:5-0:1 chloroform/methanol gradient elution, merge and obtain seven subfraction Fr.4-3-1 – Fr.4-3-7 again.Wherein Fr.4-3-5 is with anti-phase RP-18 silica gel mixed sample, and through the RP-18 column chromatography, the methanol/water gradient elution of 40:60-60:40, obtain compound R A-XII(4).The Fr.4-3-7 part is through ODSHPLC semipreparative column purifying, and 33% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain compound rubiyunnaninD(5) and rubiyunnanin E(7).The Fr.5 component is through silica gel column chromatography, and 95:5-0:1 chloroform/methanol gradient elution, merge and obtain four subfraction Fr.5-1 – Fr.5-4.Subfraction Fr.5-3 is again through silica gel column chromatography, and 20:1-0:1 ethyl acetate/methanol gradient elution, merge and obtain four subfraction Fr.5-3-1 – Fr.5-3-4.Subfraction Fr.5-3-2 and subfraction Fr.5-3-4 carry out the cyclic peptide enrichment through Sephadex LH-20 respectively, and the 1:1 chloroform/methanol is eluent.Gained contains the cyclic peptide part more respectively through anti-phase RP-18 column chromatography, and the methanol/water gradient elution of 20:80-50:50 carries out enrichment.Subfraction Fr.5-3-2 institute enrichment cyclic peptide part is through the separation and purification of ODS HPLC semipreparative column, and 23% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain compound R Y-II(11).Subfraction Fr.5-3-4 institute enrichment cyclic peptide part is through the separation and purification of ODS HPLC semipreparative column, and 40% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain compound rubiyunnanin G(9) and rubiyunnanin H(10).The Fr.6 component is through silica gel column chromatography, and 9:1:0.1-8:2:0.2 chloroform/methanol/water gradient elution, merge and obtain four subfraction Fr.6-1 – Fr.6-4.Subfraction Fr.6-3 is again through silica gel column chromatography, and 20:1-8:2 ethyl acetate/methanol gradient elution, merge the part Fr.6-3-2 that obtains containing cyclic peptide.Fr.6-3-2 is through anti-phase RP-18 column chromatography, and the methanol/water gradient elution of 10:90-40:60 obtains six subfraction Fr.6-3-2-1 – Fr.6-3-2-6.Fr.6-3-2-1 is through Sephadex LH-20 chromatography, and the 1:1 chloroform/methanol is eluent enrichment cyclic peptide, then through ODS HPLC semipreparative column purifying, 28% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain compound rubiyunnanin F(8).

NF-κ B signal pathway inhibitor take the salt of allowing on above-mentioned Rubiaceae type cyclic peptide of the present invention or its pharmacology as effective constituent.

The pharmaceutical composition that is used for the treatment of herpes simplex virus I-type, wherein contain salt and the pharmaceutically acceptable carrier of allowing on the of the present invention above-mentioned Rubiaceae type cyclic peptide for the treatment of significant quantity or its pharmacology.

the present invention also provides the Rubiaceae type cyclic peptide compounds that contains shown in the said structure formula for the treatment of significant quantity RA-V(1), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9), rubiyunnanin H(10) and RY-II(11) or the pharmaceutical composition of the salt of allowing on its pharmacology and pharmaceutically acceptable carrier.

The present invention provides the application of the salt of allowing on Rubiaceae type cyclic peptide of the present invention or its pharmacology in preparation NF-κ B signal pathway inhibitor simultaneously; And the application of the salt of allowing on Rubiaceae type cyclic peptide or its pharmacology in the medicine of preparation treatment herpes simplex virus I-type relative disease.

The present invention carries out the cyclic peptide chemical constitution study of system to Yunnan endemic species Rubiaceae Rubia plants Radix Rubiae Yunnanensis, utilize multiple separation and purification means, comprise positive reversed-phase silica gel column chromatography, Sephadex LH-20 gel chromatography, HPLC half preparations etc., therefrom obtained a series of Rubiaceae type cyclic peptide.Afterwards, the Turnover of Mouse Peritoneal Macrophages that all cyclic peptide compositions are induced at LPS and IFN-γ is to detect NO on the RAW264.7 model to generate and suppress active, detect NF-κ B path with the luciferase reporter gene method and suppress active, and it is active to detect anti-HSV-1 on the in-vitro evaluation model based on the viral HSV/Blue of restructuring report.Find wherein RA-V(1), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9), rubiyunnanin H(10) with RY-II(11) for novel NF-κ B signal pathway inhibitor; But also have the activity of good vitro inhibition herpes simplex virus I-type, can be used for preparing the medicine of anti-herpes simplex virus I type.

The salt of allowing on described Rubiaceae type cyclic peptide of the present invention or its pharmacology, can enumerate such as with the mineral acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, Hydrogen bromide, perhaps organic acids such as toxilic acid, fumaric acid, tartrate, lactic acid, citric acid, acetic acid, methylsulfonic acid, p-benzene methanesulfonic acid, hexanodioic acid, palmitinic acid, Weibull, lithium, the basic metal such as sodium, potassium, the alkaline-earth metal such as calcium, magnesium, the salt that the basic aminoacidss such as Methionin become.

The pharmaceutical composition for the treatment of herpes simplex virus I-type relative disease of the present invention, by the pharmaceutical dosage form of Rubiaceae type cyclic peptide compounds and pharmaceutically acceptable carrier preparation comprise tablet, capsule, oral liquid, injection, injection is freeze-dried or powder injection etc.Because Rubiaceae type cyclic peptide can extract separation from Radix Rubiae Yunnanensis and congener, and tablet, capsule, oral liquid, injection, injection is freeze-dried or the preparation of the pharmaceutical dosage form such as powder injection is also the conventional knowledge of this area.Therefore.Various pharmaceutical dosage forms by Rubiaceae type cyclic peptide compounds and respective carrier preparation also can be realized by those skilled in the art.

Above described pharmaceutically acceptable carrier refers to the pharmaceutical carrier of pharmaceutical field routine, such as: 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, calcium 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 add other assistant agent such as flavouring agent, sweeting agent etc. in addition in composition.

The compounds of this invention can with the form of composition by oral, snuffing enters, the mode of rectum or administered parenterally is applied to the patient who needs this treatment.Be used for can being made into conventional solid preparation such as tablet, pulvis, granula, capsule etc. when oral, make liquid preparation such as water or oil-suspending agent or other liquid preparations such as syrup, elixir etc.; While 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, then be made into required formulation.

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 age of route of administration, patient, body weight, the disease for the treatment of and severity, and its per daily dose can be 0.01～10mg/kg body weight, preferred 0.1～5mg/kg body weight.Can use by one or many.

The excellent benefit of Rubiaceae type cyclic peptide extracting method of the present invention is, at first extracts the separation thinking and has novelty, successfully utilizes the chromatography technology that anthraquinone pigment is separated with Rubiaceae type cyclic peptide.Put it briefly, utilize the molecular sieve principle of Sephadex LH-20, can separate the anthraquinone component that a part and cyclic peptide molecular weight differ greatly, recycle the adsorption chromatography principle of anti-phase RP-18 chromatographic column, the success separation obtains containing the less cyclic peptide part of pigment, then in conjunction with various chromatographic materials, comprises that the HPLC separation and purification namely obtains the cyclic peptide sterling.Secondly, only utilize the chromatographic material of laboratory or industrial routine, comprise positive reverse phase silica gel, Sephadex LH-20 and HPLC etc., needn't use special chromatographic material such as aminopropyl key and silica gel, also avoid using the chromatographic materials such as the stronger activated carbon of adsorptivity and aluminum oxide; Do not use the poor Precipitation of controllability and circulation ratio or recrystallization method, in actually operating, find that the pigment weight, be difficult to be precipitated or crystallization further separates because of the sample composition complexity; Finally, 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 Rubiaceae type cyclic peptide compounds of the present invention;

Fig. 2 is the anti-HSV-1 activity experiment of Rubiaceae type cyclic peptide compounds of the present invention;

Fig. 3 is that Rubiaceae type cyclic peptide compounds of the present invention is to the vero cytotoxicity experiment.

Embodiment:

, below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, but with this, do not limit the present invention.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:

RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9), rubiyunnanin H(10) and preparation method RY-II(11) and Structural Identification thereof Rubiaceae type cyclic peptide compounds RA-V(1):

Get the rhizome 100kg of Radix Rubiae Yunnanensis, after drying, pulverizing, with methanol eddy, extract 3 times (100L * 3 time), the time is 3h, 3h, 2h, and extracting solution obtains methanol extract 21kg through concentrating under reduced pressure.After methanol extract is added the water suspendible, fully extract with ethyl acetate and propyl carbinol successively, equal-volume respectively extracts (30L * 3 time) three times, reclaims solvent and obtains ethyl acetate part 6.4kg, propyl carbinol part 8kg and water section 5kg.Through silica gel column chromatography, with chloroform/methanol gradient elution (100:0,95:5,9:1,8:2,0:100), thin-layer chromatography, in conjunction with cyclic peptide TLC detection method merging 95:5 wherein, 9:1 and 8:1 contain the part of cyclic peptide with the ethyl acetate part.Each following step all must be carried out separation and purification in conjunction with cyclic peptide TLC detection method.Medicinal extract after merging, again through silica gel column chromatography,, with chloroform/methanol (70:1-8:2) gradient elution, is merged into six components (Fr.1-Fr.6) according to the difference of cyclic peptide point.Fr.1(139g) component is through silica gel column chromatography, and sherwood oil/acetone (7:3-0:1) obtains five subfractions (Fr.1-1 – Fr.1-5) for eluent separates.Fr.1-4(30g wherein) through Sephadex LH-20 chromatography, chloroform/methanol (1:1) is eluent, and the part that contains cyclic peptide of institute's enrichment, through silica gel H chromatography repeatedly, is separated and obtained RA-V(1 with sherwood oil/acetone (6:4) wash-out again) (4g).Fr.2(257g) component is through silica gel column chromatography, and chloroform/methanol (95:5-9:1) is carried out gradient elution, merges and obtains seven subfractions (Fr.2-1 – Fr.2-7).Fr.2-4(104g) part is with anti-phase RP-18 silica gel mixed sample, and through the RP-18 column chromatography, the methanol/water gradient elution of 20:80-90:10, obtain six subfractions (Fr.2-4-1 – Fr.2-4-6).Component Fr.2-4-2(18g) through Sephadex LH-20 chromatography, chloroform/methanol (1:1) is eluent, and the cyclic peptide part of enrichment is again through the RP-18 column chromatography, and the methanol/water gradient elution of 30:70-50:50 obtains RA-I(2) (145mg).Component Fr.2-4-5(10g) equally through Sephadex LH-20 chromatography, chloroform/methanol (1:1) is eluent, the cyclic peptide part of enrichment is through anti-phase RP-18 column chromatography, the methanol/water gradient elution of 20:80-40:60, contain cyclic peptide part again through silica gel H chromatography repeatedly, with the separation of chloroform/methanol (20:1-10:1) wash-out, obtain rubiyunnanin C(6) (34mg).Fr.3(94g) component is through silica gel column chromatography, and chloroform/methanol (70:1-9:1) is carried out gradient elution, merges and obtains five subfractions (Fr.3-1 – Fr.3-5).Fr.3-3(12g) again through silica gel column chromatography, sherwood oil/acetone (1:1-0:1) gradient elution, contain the cyclic peptide part through Sephadex LH-20 enrichment again, and chloroform/methanol (1:1) is eluent.The cyclic peptide of institute's enrichment part is through silica gel H purifying repeatedly, and chloroform/methanol (20:1) be eluent, and separation obtains RA-XXIV(3) (290mg).Fr.4(365g) component is through silica gel column chromatography, and ethyl acetate/methanol (20:1-8:2) is carried out gradient elution, merges and obtains four subfractions (Fr.4-1 – Fr.4-4).Subfraction Fr.4-3(50g) directly through Sephadex LH-20, carry out the cyclic peptide enrichment, chloroform/methanol (1:1) is eluent.Through silica gel column chromatography, chloroform/methanol (95:5-0:1) is carried out gradient elution again, merges and obtains seven subfractions (Fr.4-3-1 – Fr.4-3-7).Fr.4-3-5(21g wherein) with anti-phase RP-18 silica gel mixed sample, through the RP-18 column chromatography, the methanol/water gradient elution of 40:60-60:40, obtain RA-XII(4) (6g).Fr.4-3-7(46mg) part through ODS HPLC semipreparative column purifying, 33% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain rubiyunnanin D(5) (9mg) and rubiyunnanin E(7) (29mg).Fr.5(87g) component is through silica gel column chromatography, and chloroform/methanol (95:5-0:1) is carried out gradient elution, merges and obtains four subfractions (Fr.5-1 – Fr.5-4).Subfraction Fr.5-3(35g) again through silica gel column chromatography, ethyl acetate/methanol (20:1-0:1) is carried out gradient elution, merges and obtains four subfractions (Fr.5-3-1 – Fr.5-3-4).Subfraction Fr.5-3-2(4.5g) and subfraction Fr.5-3-4(6g) carry out the cyclic peptide enrichment through Sephadex LH-20 respectively, chloroform/methanol (1:1) is eluent.Gained contains the cyclic peptide part more respectively through anti-phase RP-18 column chromatography, and the methanol/water gradient elution of 20:80-50:50 carries out enrichment.Subfraction Fr.5-3-2 institute enrichment cyclic peptide part is through the separation and purification of ODS HPLC semipreparative column, and 23% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain compound R Y-II(11) (57mg).Subfraction Fr.5-3-4 institute enrichment cyclic peptide part is through the separation and purification of ODS HPLC semipreparative column, and 40% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain rubiyunnanin G(9) (6mg) and rubiyunnanin H(10) (8mg).Fr.6(151g) component is through silica gel column chromatography, and chloroform/methanol/water (9:1:0.1-8:2:0.2) carries out gradient elution, merges and obtains four subfractions (Fr.6-1 – Fr.6-4).Subfraction Fr.6-3(27g) again through silica gel column chromatography, ethyl acetate/methanol (20:1-8:2) is carried out gradient elution, merges the part Fr.6-3-2 that obtains containing cyclic peptide.Fr.6-3-2 is through anti-phase RP-18 column chromatography, and the methanol/water gradient elution of 10:90-40:60 obtains six subfractions (Fr.6-3-2-1 – Fr.6-3-2-6).Fr.6-3-2-1 is through Sephadex LH-20 chromatography, and chloroform/methanol (1:1) is eluent enrichment cyclic peptide, then through ODS HPLC semipreparative column purifying, 28% acetonitrile and 4 ‰ trifluoroacetic acids are eluent, obtain rubiyunnanin F(8) (22mg).

Compound rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9) and rubiyunnanin H(10) structural formula as follows:

Rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9) and rubiyunnanin H(10) the Structural Identification data be:

Rubiyunnanin D (5): white unformed powder; (c0.57, MeOH); UV (MeOH) λ _max(log ε) 203 (4.69), 276 (3.70) nm; IR (KBr) ν _max3421,2938,1659,1516,1448,1203,1138,838,722cm ^-1for ¹H and ¹³C NMR data, in Table 2; Positive FABMS m/z 801 (10) [M+H] ⁺Positive HRESIMS m/z 823.3259[M+Na] ⁺, cacld for C ₄₁H ₄₈N ₆O ₁₁Na, 823.3278.

Rubiyunnanin C (6): white crystal; Mp 253-254 ℃;

(c0.11, CHCl ₃: MeOH=1:1); UV (MeOH) λ _max(log ε) 203 (4.80), 277 (3.78) nm; IR (KBr) ν _max3414,2937,1737,1660,1514,1445,1209,1096,838,803cm ^-1for ¹H and ¹³C NMR data, in Table 2; Positive FABMS m/z 829 (100) [M+H] ⁺Positive HRFABMS m/z 828.3713[M] ⁺, cacld for C ₄₃H ₅₂N ₆O ₁₁, 828.3694.

Rubiyunnanin E (7): white unformed powder;

(c0.58, MeOH); UV (MeOH) λ _max(log ε) 204 (4.81), 280 (3.87) nm; IR (KBr) ν _max3426,2938,1664,1515,1446,1204,1134,839,801,722cm ^-1for ¹H and ¹³C NMR data, in Table 2; Positive FABMS m/z 831 (50) [M+H] ⁺Positive HRESIMS m/z 853.3391[M+Na] ⁺, cacld for C ₄₂H ₅₀N ₆O ₁₂Na, 853.3384.

Rubiyunnanin F (8): white unformed powder;

(c0.37, MeOH); UV (MeOH) λ _max(log ε) 203 (4.87), 276 (3.80) nm; IR (KBr) ν _max3423,2932,1663,1512,1447,1414,1248,1208,1075,839,803cm ^-1for ¹H and ¹³C NMR data, in Table 3; Positive FABMS m/z 976 (92) [M+H] ⁺Positive HRESIMS m/z 976.4334[M+H] ⁺, cacld for C ₄₈H ₆₂N ₇O ₁₅, 976.4303.

Rubiyunnanin G (9): white unformed powder; (c0.11, MeOH); UV (MeOH) λ _max(log ε) 203 (4.57), 277 (3.53) nm; IR (KBr) ν _max3440,2936,1658,1640,1517,1449,1207,838,810cm ^-1for ¹H and ¹³C NMR data, in Table 3; Positive FABMS m/z 905 (24) [M+H] ⁺Positive HRESIMS m/z 927.3734[M+Na] ⁺, cacld for C ₄₅H ₅₆N ₆O ₁₄Na, 927.3752.

Rubiyunnanin H (10): white unformed powder;

(c0.12, MeOH); UV (MeOH) λ _max(log ε) 203 (4.81), 276 (3.83) nm; IR (KBr) ν _max3424,2933,1657,1640,1512,1248,1075cm ^-1for ¹H and ¹³C NMR data, in Table 3; Positive FABMS m/z 935 (95) [M+H] ⁺Positive HRESIMS m/z 957.3882[M+Na] ⁺, cacld for C ₄₆H ₅₈N ₆O ₁₅Na, 957.3857.

Table 1rubiyunnanin D(5), rubiyunnanin C(6) and rubiyunnanin E(7) ¹H NMR and ¹³C NMR data (C ₅D ₅N, δ in ppm, J in Hz)

^a?Data?were?measuredat125MHz. ^b?Data?were?measured?at500MHz.

Table 2rubiyunnanin F(8), rubiyunnanin G(9) and rubiyunnanin H(10) ¹H NMR and ¹³C NMR data (CD ₃OD, δ in ppm, J in Hz)

^a?Data?were?measured?at100MHz. ^b?Data?were?measured?at500MHz. ^c?Data?were?measured?at400MHz.

Embodiment 2:

The Turnover of Mouse Peritoneal Macrophages of Rubiaceae type cyclic peptide compounds RA-V(1 of the present invention), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnaninF(8), rubiyunnanin G(9), rubiyunnanin H(10) and RY-II(11) at LPS and IFN-γ, inducing is can obviously suppress NO on the RAW264.7 model to generate.Experimental principle, method and result are as follows:

Experimental principle: coding nitric oxide synthetase (Nitric Oxide Synthase, NOS) gene is one of direct target gene of NF-κ B signal path, can activate NF-κ B signal path while suffering the stimulations such as microorganism intracellular toxin, inflammatory mediator when cell, thereby generate a large amount of induced NOSs, nitricoxide synthase is by its substrate L-arginine of catalysis, generate NO and Cit, NO form with nitrite ion in the aqueous solution exists.This experimental applications LPS and IFN-γ stimulate the RAW264.7 cell, then apply Griess reagent and measure NO in cell culture supernatant ₂ ^-Level is lived with the overall enzyme of reflection induced NOS.

Experimental technique: (1) inoculating cell: with the nutrient solution that contains 10% foetal calf serum, the RAW264.7 cell is made into the individual cells suspension, with every hole 2 * 10 ⁵Individual cell is inoculated into 96 orifice plates.(2) compound treatment: after cell attachment, add LPS and IFN-γ to induce nitrogen protoxide to generate, add simultaneously testing compound solution, continue to cultivate 24 hours.(3) colour developing: draw cell conditioned medium liquid and add the Griess reagent colour development, 570nm detects absorbancy.Measure the cell survival amount with batch cell application mtt assay.(4) record result, take concentration as X-coordinate, cell NO growing amount and survival rate are the ordinate zou curve plotting, the IC of application Reed and Muench method computerized compound ₅₀Value.

Experimental result:

The half-inhibition concentration that table 3 Rubiaceae type cyclic peptide 1-11 generates NO

Compound	1	2	3	4	5	6	7	8	9	10	11	MG-132
													IC ₅₀（μM）	0.05	0.25	2.22	0.32	7.22	5.16	11.25	1.77	12.64	12.68	1.99	0.35

Experimental result shows, compound 1-11 all have suppress the activity that NO generates, wherein RA-V(1), RA-I(2) and RA-XII(4) activity be better than positive control proteasome inhibitor MG-132, RA-V(1) activity is the strongest, IC ₅₀Value reaches 50nM.

Embodiment 3:

Rubiaceae type cyclic peptide compounds RA-V(1 of the present invention), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9), rubiyunnanin H(10) and RY-II(11) with the luciferase reporter gene method, detect NF-κ B path and suppress active, further prove conclusively the cyclic peptide molecule and really act on NF-κ B path.Experimental principle, method and result are as follows:

Experimental principle: NF-κ B nuclear factor has conservative DNA binding sequence row, by with its combination, the expression of regulation and control downstream gene.PNF-κ B-Luc reporter plasmid (Clontech) is loaded with the Photinus pyralis LUC reporter gene, the expression of this gene is regulated and controled by synthetic promotor, wherein comprise basic promoter element (TATA box), and the DNA binding sequence of NF-κ B transcription factor row, so the expression of Photinus pyralis LUC reporter gene is regulated and controled by NF-κ B signal path.Can reflect the regulating and controlling effect of compound to NF-κ B signal path by investigating compound to luciferase reporter gene expression variation.

Experimental technique: the HEK293 cell of (1) inoculation pNF-κ B-Luc stable transfection: with the HG-DMEM nutrient solution that contains 10% foetal calf serum, the HEK293 cell is made into the individual cells suspension, with every hole 1 * 10 ⁴Individual cell is inoculated into 96 orifice plates.(2) compound treatment: after cell cultures 24h, first add testing compound solution (compound from 25 μ g/mL to 0.1 μ g/mL, 3 times of dilutions), cultivate 1h, then add 10ng/mL TNF-α to induce NF-κ B signal path to activate, continue to cultivate 2h.(3) luciferase assays: luciferase assays adopts single substrate luciferase detection kit (Promega of Promega company, E2510), add the single substrate luciferase of 50 μ L substrate, lucifuge is hatched 30min, at Envision multiple labeling microwell plate detector (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA) on the value of reading analyze.Measure the cell survival amount with batch cell application mtt assay.(4) record result, take independent TNF-α treatment group as 100%, the original value of reading homogenization is processed, take concentration as X-coordinate, luciferase signal relative value is the ordinate zou curve plotting, the IC50 value of computerized compound.

Experimental result:

The restraining effect of table 4 Rubiaceae type cyclic peptide compounds 1-11 to NF-κ B signal path

Compound	1	2	3	4	5	6	7	8	9	10	11	PS-341
													IC ₅₀（μM）	0.03	1.69	12.64	1.18	35.07	ND	ND	ND	ND	ND	ND	0.14

ND:no?detected(>25μg/mL)

Experimental result shows, it is active that compound 1-11 all has the inhibition of NF-κ B signal path, wherein RA-V(1) activity is the strongest, IC ₅₀For 30nM, it is suitable that its activity is better than the positive control synthetic compound PS341 order of magnitude, greatly is better than most of NF-κ B signal path natural inhibitors of present discovery.So Rubiaceae type cyclic peptide is the NF-κ B signal path natural inhibitor of a class formation novelty.

Embodiment 4:

Rubiaceae type cyclic peptide compounds RA-V(1 of the present invention), RA-I(2), RA-XXIV(3), RA-XII(4), rubiyunnanin D(5), rubiyunnanin C(6), rubiyunnanin E(7), rubiyunnanin F(8), rubiyunnanin G(9), rubiyunnanin H(10) and the activity of certain anti-herpes simplex virus I type is arranged RY-II(11), compound R A-V(1 wherein) and RA-XII(4) have an activity of anti-herpes simplex virus I type preferably.Anti-herpes simplex virus I type experimental principle, method and result are as follows:

Experimental principle: the anti-HSV-1 activation evaluating system that uses is a kind of in-vitro evaluation system based on the viral HSV/Blue of restructuring report.Viral load in recombinant virus HSV/Blue infected cell is directly proportional within the specific limits with the quantity that reporter gene lacZ expresses, but the activity that therefore detects lacZ expression product β-Gal in its linearity range indirect detection to viral quantity.Reporter gene activity by comparison process group and virus control group in experiment, thus the anti-HSV-1 of assess sample is active.

Experimental technique:

(1) cytotoxicity of sample detects

Vero cell (African green monkey kidney cell) is cultivated and is formed individual layer in 96 porocyte culture plates after, the sample liquid that adds different concns, after continuing to cultivate 48h, the nutrient solution that replacing contains 10%Alamarblue continue to be cultivated after 4h the fluorescent value that detects its 530/590nm place, the toxicity of test sample to the Vero cell, and calculate half cell toxicant concentration (CC ₅₀).

(2) the anti-HSV-1 virus function of sample detects

The Vero cell is cultivated and is formed individual layer in 96 porocyte culture plates after, 0.5M virus liquid and be no more than 20% Cytotoxic gradient concentration sample solution and be added to simultaneously on the Vero cell, after continuing to cultivate 24h, expression product β-Gal of examining report gene lacZ, determine the restraining effect of sample to HSV-1, and calculation of half inhibitory concentration (IC ₅₀).

Experimental result:

The anti-HSV-1 of table 5 Rubiaceae type cyclic peptide compounds 1-11 is active

Experimental result shows, it is active that compound 1-11 has certain anti-HSV-1.Wherein the anti-HSV-1 of RA-V is significantly active, and selectivity index (SI) reaches 22.9.

Embodiment 5:

Embodiment 1 gained compound 1-11, add 4% ethanol solution of sulfuric acid, and PH=4 filters, and drying, make sulphate cpd 1-11.

Embodiment 6:

Embodiment 1 gained compound 1-11, add 4% hydrochloric acid soln, and PH=4 filters, and drying, make hydrochloride compound 1-11.

Embodiment 7:

Embodiment 1 gained compound 1-11, add 4% tartaric acid solution, and PH=4 filters, and drying, make tartrate compound 1-11.

Embodiment 8:

Embodiment 1 gained compound 1-11, add 4% citric acid solution, and PH=4 filters, and drying, make Citrate trianion compound 1-11.

Embodiment 9:

The salt 10mg of tablet: embodiment 1 gained compound 1-11 or embodiment 5-8 gained, lactose 180mg, starch 55mg, Magnesium Stearate 5mg.

The preparation method: compound or its salt, lactose and starch is mixed, water evenly moistening, the mixture after moistening is sieved and dry,, after sieve, add Magnesium Stearate, then with the mixture compressing tablet, every weight 250mg, compounds content is 10mg.

Embodiment 10:

The salt 2mg of ampulla: embodiment 1 gained compound 1-11 or embodiment 5-8 gained, sodium-chlor 10mg.

Preparation method: compound or its salt and sodium-chlor are dissolved in appropriate water for injection, filter gained solution, in the ampoule of packing under aseptic condition.

Embodiment 11:

Injection is freeze-dried: the salt 10mg of embodiment 1 gained compound 1-11 or embodiment 5-8 gained, sodium bicarbonate 2mg, N.F,USP MANNITOL 252mg.

Preparation method:, with sodium bicarbonate, N.F,USP MANNITOL, be dissolved in water for injection, add activated carbon adsorption 30min depyrogenation, remove by filter activated carbon, add compound or its salt in filtrate, supersound process makes dissolving, regulating PH with 1N hydrochloric acid is 5.0-7.0, millipore filtration filters, and injects water, packing, lyophilize, top plug, roll lid, obtains.

Embodiment 12:

The salt 10mg of capsule: embodiment 1 gained compound 1-11 or embodiment 5-8 gained, lactose 187mg, Magnesium Stearate 3mg.

The preparation method: compound or its salt and solubility promoter is mixed, sieve, evenly mix, the mixture that the obtains hard gelatin capsule of packing into, each capsule weight 200mg, active component content is 10mg.

Claims

1. the Rubiaceae type cyclic peptide compounds rubiyunnanin F(8 shown in following structural formula),

2. the pharmaceutical composition that is used for the treatment of herpes simplex virus I-type, wherein contain salt and the pharmaceutically acceptable carrier of allowing on the Rubiaceae type cyclic peptide of the claim 1 for the treatment of significant quantity or its pharmacology.

3. the application of the salt of allowing on claim 1 described Rubiaceae type cyclic peptide or its pharmacology in the medicine of preparation treatment herpes simplex virus I-type relative disease.