CN101993474A - Glaucocalyxin A derivatives with cyclic octapeptide structure as well as preparation method and application of glaucocalyxin A derivatives - Google Patents
Glaucocalyxin A derivatives with cyclic octapeptide structure as well as preparation method and application of glaucocalyxin A derivatives Download PDFInfo
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Abstract
The invention discloses a structure of glaucocalyxin A derivatives with cyclic octapeptide structure as well as a preparation method and application of the glaucocalyxin A derivatives in treating cancer. The general formula of the molecular structure of the glaucocalyxin A derivative with cyclic octapeptide structure is shown in the specification, wherein R1 and R2 are -OH or a group shown in the specification, and R3 is a cyclic octapeptide substituent group. The preparation method comprises the following steps: in the presence of tetrahydrofuran and 4-dimethylin pyridine, reacting extracted glaucocalyxin A with succinic acid; in the presence of tetrahydrofuran and dichloroethane, reacting the obtained succinate of glaucocalyxin A with N-hydroxyl succinimide to obtain the intermediate product; and finally, in the presence of tetrahydrofuran and phosphate buffer, carrying out derivatization reaction on the obtained intermediate product with cyclic octapeptide, thereby obtaining the glaucocalyxin A derivatives with the cyclic octapeptide structure.
Description
Technical field
The present invention relates to glaucocalyxin A (GLA) derivative, the glaucocalyxin A derivative that particularly has the cyclic octapeptide structure.The invention still further relates to described preparation method and the application in the treatment cancer thereof with glaucocalyxin A derivative of cyclic octapeptide structure.
Background technology
Labiatae Rabdosia plant.Widely distribute at east Asia and African western part, the whole world has 150 kinds approximately, and China has 90 kinds of 25 mutation approximately.Wherein have approximately 30 kinds among the people as medicinal, as clearing heat and detoxicating, anticancer anti-inflammatory, invigorating the spleen, invigorate blood circulation, Hangzhoupro bacterium medicine uses.People to this platymiscium-the mushroom constituents done research, therefrom extracted over one hundred kind of medicine constituents.Find that through pharmacological screening many diterpene compounds have cell toxicant, antitumor, Hang Yan active function.
Rabdosia japonica is a Labiatae Rabdosia plant, is distributed in northeast, the North China of China, Korea. Japan, and former Soviet Union the Far East Area, Jilin Province's resource is especially abundant.Rabdosia japonica have be good for the stomach, clearing heat and detoxicating, invigorate blood circulation, antisepsis and anti-inflammation and antitumour activity, be used for gastritis, hepatitis from the beginning of diseases such as, cold, fever, mazoitis, arthrodynia.Modern study finds that herb has certain effect to cardiovascular, and effective constituent wherein has certain influence to platelet aggregation and cancer.
People such as Yunlong extracts glaucocalyxin A (glaucocalyxinA) and Glaucocalyxin B (glaucocalyxinB) about 1981 from rabdosia japonica; infer that from spectrum glaucocalyxin A has a typical skilful oxygen-16 1 kaurene (ent-15-oxo-l6-kaurene) skeleton of Rabdosia diterpene; and Glaucocalyxin B is 14 1 acetylates of glaucocalyxin A; both are distinguished acetylize; obtain identical acetylate, thereby confirmed both mutual relationships.Become also from the product rabdosia japonica of Jilin, to get this two kinds of diterpene with the later Chao Dynasty is complete.
Liu Chen rivers in 1988 etc. are produced from the Beijing area and are got the rabdosia japonica outside first element and the second element, also get blue calyx third element (glaucocalyxinC), and structure is mapping-7p, 14a, 15a-trihydroxy--16-kauri pine-3-ketone.Dongsa kimls, except therefrom telling glaucocalyxin A, Glaucocalyxin B and blue calyx third element, also told two kinds of new diterpene glaucocalyxinD and glaucocalyxinE and listed the structure infrared and ultraviolet absorption value of these five kinds of compounds, topmost 1H one nucleus magnetic resonance, 13C one chemical shift of NMR value and mass-spectrometric data value.
Kim Yong Ils etc. have been got glaucocalyxin A from the rabdosia japonica root, king elder generation honor etc. is produced the Isodon amethystoides (c) (Isodonamethystoides (Benth) CyWuetHsuan) from Anhui and also got glaucocalyxin A in addition.Human reversed-phased high performace liquid chromatographic external standard methods such as Wang Huifen are measured the content of glaucocalyxin A in different sites and the rabdosia japonica of different acquisition phase, and the result shows that glaucocalyxin A is far above root, stem in the leaf.The different acquisition phase is with 7-8 month content height, 9 the end of month content descend.Zhang Yuantong etc. also adopt reversed-phase HPLC to measure glaucocalyxin A content, and recording glaucocalyxin A content is 1.03%, and average recovery rate is 99.2%.
From glaucocalyxin A experiment in vivo and vitro result, it has stronger antitumor action, and antitumor spectra is wider, can suppress the growth of solid tumors such as Lewis lung cancer, 5180 solid-types and HCA solid-type, obviously increase the increase in life span of lotus ascitic type 5180 ascitic types and lotus HCA ascitic type mouse, the power of its antitumor action is dose-dependently.
The scientific research personnel has carried out the Anticancer Activities of human promyelocytic leukemia cell, Proliferation of Human Ovarian Cell and human lung carcinoma cell with mtt assay to 14 the known mappings-kaurene class diterpene compound of isolation identification from hair leaf Herba Rabdosiae glaucocalycis leaf, and the compound that wherein has an alpha-methylene cyclopentanone structure all shows in various degree inhibition activity to above-mentioned 3 kinds of tumor cell lines.From rabdosia japonica leaf isolated compound such as blue honor Amethystoidin A (GLA) and second element (GLB) structure, have the alpha-methylene cyclopentanone, thereby demonstrate stronger biological activity.
The anti tumor activity in vitro screening and the structure activity study that Bake China fir vinyl compound after some natural and chemically modifieds have been carried out system show, alpha-methylene cyclopentanone structural unit among the applied chemistry means saboteur, the significantly anti-tumor activity of reduction Bake China fir vinyl compound.
Because glaucocalyxin A is mapping-kaurene class diterpene compound, its compound polarity is little, is soluble in non-polar solvents such as chloroform, and is insoluble in water, therefore is not suitable for directly carrying out administration as medicine; Glaucocalyxin A is external to have tangible antitumous effect, but needs heavy dose of long-time medication could produce drug effect in vivo.Medicine is eliminated very fast in vivo, and the transformation period is short, still can not be directly as drug use.
Summary of the invention
In view of glaucocalyxin A (GLA) particularly its alpha-methylene cyclopentanone structure the tumor cell line activity is had good inhibition effect, yet because characteristics such as its polarity is little, transformation period weak point, it is unsuitable for directly carrying out administration as medicine, so need be lead compound with glaucocalyxin A (GLA) now, by structure of modification to lead compound, obtain the derivative of glaucocalyxin A, thereby make it according to known pathways metabolism inactivation or not metabolism, and excrete by original shape, thereby improve the security of medicine, increase drug effect.
One of purpose of the present invention is to provide has the glaucocalyxin A derivative that has the cyclic octapeptide structure to the tumor cell line good inhibition effect, structure improved, GLA polarity is little, not soluble in water, the transformation period short, eliminate defectives such as too fast in vivo thereby solve, thereby as the drug use for the treatment of cancer.
Two of purpose of the present invention is to provide described preparation method with glaucocalyxin A derivative of cyclic octapeptide structure, the bigger defective of toxic side effect of avoiding simple employing organic synthesis to be brought, and the drug effect of avoiding simple employing feedstock purification to bring lower more, can't specific aim treat the defective of a certain particular cancers.
Glaucocalyxin A derivative with cyclic octapeptide structure disclosed in this invention is a kind of kaurane type tetracyclic diterpene class material, has kaurane type structure, carbonyl, hydroxyl and cyclic octapeptide structure, and the general formula of its molecular structure is as follows:
Wherein, R1, R2 be-OH or
And R3 is the cyclic octapeptide substituting group.
In a preferred embodiment, there is one to be-OH among described R1 and the R2.
In a preferred embodiment, described R3 is a cyclic octapeptide
Substituting group
Described cyclic octapeptide has D type or L type structure, and the molecular formula of wherein said cyclic octapeptide is C33H48N11O17S2.
Studies show that above-mentioned cNGQGEQc cyclic octapeptide has specificity and combines with nonsmall-cell lung cancer (A-549), lung squamous cancer Calu-1, lung adenosquamous carcinoma H178 cell, this is to finish by the mediation of integrin alpha 3.
So introduced above-mentioned cyclic octapeptide substituting group in the glaucocalyxin A, make GLA derivative after modified to carry out specificity and combine with corresponding tumour cell, promptly can carry out target administration, thereby bring into play effective antitumous effect tumour cell.
In a preferred embodiment, described R1 is
Described R2 is-OH that described R3 is a D type cyclic octapeptide substituting group
Its molecular structure is as follows:
In a preferred embodiment, described R2 is
Described R1 is-OH that described R3 is a D type cyclic octapeptide substituting group
Its molecular structure is as follows:
In a preferred embodiment, described R1 is
Described R2 is-OH that described R3 is a L type cyclic octapeptide substituting group
Its molecular structure is as follows:
In a preferred embodiment, described R2 is
Described R1 is-OH that described R3 is a L type cyclic octapeptide substituting group
Its molecular structure is as follows:
Above-mentioned have the glaucocalyxin A derivative of cyclic octapeptide structure owing to have the alpha-methylene cyclopentanone structure of GLA, so it has good inhibition effect to the tumor cell line activity, on the other hand, above-mentioned have the glaucocalyxin A derivative of cyclic octapeptide structure owing to have the structural modification of cyclic octapeptide, so it has the specificity combination to tumor cell line, thereby tumor cell line is carried out target administration, and above-mentioned glaucocalyxin A derivative with cyclic octapeptide structure has carried out structural modification, it is little to have solved polarity, defective characteristics such as the transformation period is short are suitable for the drug use as the treatment cancer.
Above-mentioned glaucocalyxin A derivative with cyclic octapeptide structure is prepared in accordance with the following methods, and it comprises and is used to prepare as step 1～4 of the glaucocalyxin A of lead compound and is used for the step 5 that the structural modification glaucocalyxin A carries out derivatize.
Described step 1 is an extraction step, specifically may further comprise the steps:
Step 1.1: get Herba Rabdosiae glaucocalycis medicinal material (over-ground part) and be crushed to 20 orders～50 orders;
Step 1.2: the crushed material of gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 5～1: 15, and heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, and obtained extracting solution and residuum;
Step 1.3: the residuum of step 1.2 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 5～1: 15, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, obtain extracting solution and residuum, repeat this step 1～3 time;
Step 1.4: the extracting solution of combining step 1.2 and 1.3 gained.
Described step 2 is the solvent treatment step, specifically may further comprise the steps:
Step 2.1: with the extracting solution heating of step 1 gained, under 55 ℃～65 ℃ temperature condition, heat concentrating under reduced pressure, reclaim ethanol and obtain the elementary enriched material of heavy-gravity;
Step 2.2: the elementary enriched material of step 2.1 gained was mixed according to volume ratio with water in 1: 8～1: 10, stirred 10 minutes with 60～120 rev/mins speed at ambient temperature, left standstill subsequently 6～12 hours, abandoning supernatant obtains lower floor's solid;
Step 2.3: the solid and the ethyl acetate (A.R.) of step 2.2 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition,, left standstill 1～3 hour, filter and obtain filtrate and insolubles with 60～120 rev/mins speed stirring and dissolving;
Step 2.4: the insolubles and the ethyl acetate (A.R.) of step 2.3 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition with 60～120 rev/mins speed stirring and dissolving, left standstill 1～3 hour, and filtered and to obtain filtrate and insolubles, repeat this step 1～3 time;
Step 2.5: the filtrate of combining step 2.3 and 2.4 gained;
Step 2.6: with the filtrate heating of step 2.5 gained, under 35 ℃～45 ℃ temperature condition, heat concentrating under reduced pressure, reclaim ethyl acetate (A.R.), obtain solid concentrates.
Described step 3 is a resin treatment step, specifically may further comprise the steps:
Step 3.1: the solid concentrates of step 2 gained and 95% ethanol (A.R.) according to 1: 4～1: 6 mixed dissolution of volume ratio, are obtained solution;
Step 3.2: the solution of step 3.1 gained slowly is added in No. 7 resin columns, work the color of observing resin, when resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 200ml～400ml to wash this resin column, collect all effluent liquid, the weighting material of described resin column is a strongly basic anionic resin and to be saturated to the pH value with sodium hydroxide neutral;
Step 3.3: with the heating of step 3.2 gained effluent liquid, and under 55 ℃～65 ℃ temperature condition, heat reduced-pressure backflow, do, obtain solid residue until solvent.
Described step 3 is a re-crystallization step, specifically may further comprise the steps:
Step 4.1: the polar solvent of above-mentioned residue with 30 ℃～40 ℃ mixed according to volume ratio in 1: 4～1: 6, obtain primary solution, heating, 30 ℃～40 ℃ of temperature condition under concentrating under reduced pressure, under-23 ℃～-13 ℃ temperature condition, leave standstill crystallization, filter and obtain light yellow needle crystal;
Described polar solvent is the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.);
Step 4.2: the crystallization of step 4.1 gained was mixed according to volume ratio with polar solvent in 1: 4～1: 6, and recrystallization 2～4 times repeatedly under-23 ℃～-13 ℃ temperature condition each 12～24 hours, obtains the intermediate product glaucocalyxin A;
Described polar solvent is the mixing solutions according to 1: 1～1: 4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Described step 5 is the derivatize step, specifically may further comprise the steps:
Step 5.1: at room temperature, 9.06～11.06g step 4 gained is dissolved in glaucocalyxin A in 25～35ml tetrahydrofuran (THF) (THF), add 0.857～1.057g Succinic Acid and 0.067～0.08794-Dimethylamino pyridine (DMAP) subsequently, stirring reaction is 2～3 hours at ambient temperature;
Step 5.2: with 30 ℃～40 ℃ concentrating under reduced pressure of reaction soln heating of step 5.1, remove THF, add the dilution of 25～35ml water subsequently, and adopt 25～35ml ethyl acetate extraction 3 times, merge organic phase and adopt saturated common salt water washing, anhydrous sodium sulfate drying, subsequently gained solution is heated to 30 ℃～40 ℃ and is evaporated to driedly, obtain the thick product of the succinate of glaucocalyxin A;
Step 5.3: adopt silica gel column chromatography to separate the thick product of the succinate of glaucocalyxin A, described elutriant is that volume ratio is 1: 1 a petroleum ether-ethyl acetate mixing solutions, adopt the elutriant flushing chromatography column of 2 times of column volumes (2BV), then the thick product of the succinate of described glaucocalyxin A is dissolved the back fully with elutriant and add pillar, adopt the elutriant flushing chromatography column of 4BV again, collect and merge the cut that contains target component, the gained cut is heated to 30 ℃～40 ℃ concentrating under reduced pressure, remove and desolvate, obtain the clear crystal of the succinate of glaucocalyxin A;
Step 5.4: the succinate of gained glaucocalyxin A is dissolved in 15～20ml tetrahydrofuran (THF) (THF), at room temperature, adds 0.5～0.6g ethylene dichloride (EDC) and 0.2～0.3gN-N-Hydroxysuccinimide (NHS), stirring at room 8～10 hours;
Step 5.5: with the underpressure distillation under 30 ℃～40 ℃ conditions of gained reaction soln, remove THF, adopt 15～20ml washed with dichloromethane subsequently 3 times, use saturated common salt water washing organic phase again, anhydrous sodium sulfate drying filters and with the underpressure distillation under 30 ℃～40 ℃ conditions of gained filtrate, removes and desolvate, dry at ambient temperature, the clear crystal of derivative of the succinate of glaucocalyxin A and NHS;
Step 5.6: at ambient temperature, the glaucocalyxin A derivative of 55～65mg step 5.5 is dissolved in 0.9～1.1mlTHF solution, and with 58～68mg cyclic octapeptide
Be dissolved in 4～6mlPBS buffered soln, the THF drips of solution with the glaucocalyxin A derivative is added in the PBS solution of cyclic octapeptide subsequently, and stirring reaction is 10～20 minutes under the room temperature condition;
Step 5.7: THF is removed in reaction soln underpressure distillation under 30 ℃～40 ℃ conditions of step 5.6, subsequent filtration also adopts 1.5～2.5ml water washing 3 times with gained filtrate, merging filtrate, and lyophilize 20～28 hours under-45 ℃～-35 ℃ conditions, the glaucocalyxin A derivative that obtains having the cyclic octapeptide structure.
Glaucocalyxin A derivative through above-mentioned preparation process gained with cyclic octapeptide structure, owing to have alpha-methylene cyclopentanone structure and cyclic octapeptide fragment, so it has good inhibition effect and targeting to tumor cell line, can be used for treating cancer, be used in particular for treating cancer of the stomach, prostate gland, leukemia, nasopharyngeal carcinoma, liver cancer, pancreas cholangiocarcinoma, ovarian cancer, mammary cancer, the esophageal carcinoma, lung cancer, kidney, cervical cancer, colorectal carcinoma.
The present invention discloses the described application of glaucocalyxin A derivative in the treatment cancer, the particularly application in treatment cancer of the stomach, prostate gland, leukemia, nasopharyngeal carcinoma, liver cancer, pancreas cholangiocarcinoma, ovarian cancer, mammary cancer, the esophageal carcinoma, lung cancer, kidney, cervical cancer, colorectal carcinoma with cyclic octapeptide structure.
The glaucocalyxin A derivative with cyclic octapeptide structure of aforementioned arbitrary structure disclosed in this invention can adopt the conventional medicine carrier on the pharmaceutics to make any formulation, includes but not limited to tablet, capsule, flexible glue agent, sprays, gelifying agent, gel inhalation, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection, infusion solution, freeze dried injection, lipidosome injection, target administration injection, suppository, sustained release preparation or controlled release preparation.
Description of drawings
Fig. 1 a is the reaction equation of glaucocalyxin A and Succinic Acid.
Fig. 1 b is the reaction equation of the succinate and the N-hydroxy-succinamide of glaucocalyxin A.
Fig. 2 a is succinate and one of the derivative of NHS and reaction equation of D type cyclic octapeptide of glaucocalyxin A.
Fig. 2 b be the derivative of the succinate of glaucocalyxin A and NHS and D type cyclic octapeptide reaction equation two.
Fig. 3 a has the HPLC figure of the glaucocalyxin A derivative of D type cyclic octapeptide structure for Fig. 2 a～2b shows the reaction gained.
Fig. 3 b has the mass spectrum of the glaucocalyxin A derivative of D type cyclic octapeptide structure for Fig. 2 a～2b shows the reaction gained.
Fig. 4 a is succinate and one of the derivative of NHS and reaction equation of L type cyclic octapeptide of glaucocalyxin A.
Fig. 4 b be the derivative of the succinate of glaucocalyxin A and NHS and L type cyclic octapeptide reaction equation two.
Fig. 5 a has the HPLC figure of the glaucocalyxin A derivative of L type cyclic octapeptide structure for Fig. 4 a～4b shows the reaction gained.
Fig. 5 b has the mass spectrum of the glaucocalyxin A derivative of L type cyclic octapeptide structure for Fig. 4 a～4b shows the reaction gained.
Embodiment
According to claim of the present invention and the disclosed content of summary of the invention, technical scheme of the present invention specifically as described in the following Examples.
Preparation method disclosed in this invention is under the condition of tetrahydrofuran (THF) (THF) and 4-Dimethylamino pyridine (DMAP) existence, will be through extracting glaucocalyxin A and the Succinic Acid reaction after handling, under the condition that tetrahydrofuran (THF) (THF) and ethylene dichloride (EDC) exist, the succinate of the glaucocalyxin A that obtains reacted with N-hydroxy-succinamide (NHS) subsequently and obtain intermediate product, at last under the condition of tetrahydrofuran (THF) (THF) and phosphate buffered saline buffer (PBS damping fluid) existence, gained intermediate product and cyclic octapeptide are carried out derivative reaction, thereby prepare the glaucocalyxin A derivative with cyclic octapeptide structure disclosed in this invention, this preparation process mainly comprises the preparation and the substituent derivatize of glaucocalyxin A.The described preparation process of following examples, the various chemical reagent that adopted then are analytical pure as there not being special mark.
Embodiment 1: the preparation of glaucocalyxin A
Adopt Herba Rabdosiae glaucocalycis medicinal material (over-ground part) as raw material, by steps such as extraction, solvent treatment, plastic resin treatment and recrystallizations, prepare glaucocalyxin A (GLA), its detailed process is as follows:
Step 1 is an extraction step, and it further comprises:
Step 1.1: get Herba Rabdosiae glaucocalycis medicinal material (over-ground part) and be crushed to 20 orders～50 orders;
Step 1.2: the crushed material of gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 5～1: 15, and heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, and obtained extracting solution and residuum;
Step 1.3: the residuum of step 1.2 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 5～1: 15, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, obtain extracting solution and residuum, repeat this step 1～3 time;
Step 1.4: the extracting solution of combining step 1.2 and 1.3 gained.
Step 2 is the solvent treatment step, and it further comprises:
Step 2.1: with the extracting solution heating of step 1 gained, under 55 ℃～65 ℃ temperature condition, heat concentrating under reduced pressure, reclaim ethanol and obtain the elementary enriched material of heavy-gravity;
Step 2.2: the elementary enriched material of step 2.1 gained was mixed according to volume ratio with water in 1: 8～1: 10, stirred 10 minutes with 60～120 rev/mins speed at ambient temperature, left standstill subsequently 6～12 hours, abandoning supernatant obtains lower floor's solid;
Step 2.3: the solid and the ethyl acetate (A.R.) of step 2.2 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition,, left standstill 1～3 hour, filter and obtain filtrate and insolubles with 60～120 rev/mins speed stirring and dissolving;
Step 2.4: the insolubles and the ethyl acetate (A.R.) of step 2.3 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition with 60～120 rev/mins speed stirring and dissolving, left standstill 1～3 hour, and filtered and to obtain filtrate and insolubles, repeat this step 1～3 time;
Step 2.5: the filtrate of combining step 2.3 and 2.4 gained;
Step 2.6: with the filtrate heating of step 2.5 gained, under 35 ℃～45 ℃ temperature condition, heat concentrating under reduced pressure, reclaim ethyl acetate (A.R.), obtain solid concentrates.
Step 3 is a resin treatment step, and it further comprises:
Step 3.1: the solid concentrates of step 2 gained and 95% ethanol (A.R.) according to 1: 4～1: 6 mixed dissolution of volume ratio, are obtained solution;
Step 3.2: the solution of step 3.1 gained slowly is added in No. 7 resin columns, work the color of observing resin, when resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 200ml～400ml to wash this resin column, collect all effluent liquid, the weighting material of described resin column is a strongly basic anionic resin and to be saturated to the pH value with sodium hydroxide neutral;
Step 3.3: with the heating of step 3.2 gained effluent liquid, and under 55 ℃～65 ℃ temperature condition, heat reduced-pressure backflow, do, obtain solid residue until solvent.
Step 4.1: the polar solvent of above-mentioned residue with 30 ℃～40 ℃ mixed according to volume ratio in 1: 4～1: 6, obtain primary solution, heating, 30 ℃～40 ℃ of temperature condition under concentrating under reduced pressure, under-23 ℃～-13 ℃ temperature condition, leave standstill crystallization, filter and obtain light yellow needle crystal;
Described polar solvent is the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.);
Step 4.2: the crystallization of step 4.1 gained was mixed according to volume ratio with polar solvent in 1: 4～1: 6, and recrystallization 2～4 times repeatedly under-23 ℃～-13 ℃ temperature condition each 12～24 hours, obtains the intermediate product glaucocalyxin A;
Described polar solvent is the mixing solutions according to 1: 1～1: 4 blended chloroform (A.R.) of volume ratio and acetone (A.R.).
Prepare glaucocalyxin A by above-mentioned steps.
Embodiment 2: derivatize
According to the reaction equation shown in Fig. 1 a～1b, with embodiment 1 preparation-obtained glaucocalyxin A, adopt Succinic Acid and NHS to carry out derivatize, obtain intermediate product, subsequently according to the reaction equation shown in Fig. 2 a～2b and Fig. 4 a～4b, with gained intermediate product and cyclic octapeptide
Carry out derivatize, modify substituting group thereby in glaucocalyxin A, introduce, the glaucocalyxin A derivative that obtains having the cyclic octapeptide structure, described derivatize step 5 further comprises:
Step 5.1: at room temperature, 9.06～11.06g step 4 gained is dissolved in glaucocalyxin A in 25～35ml tetrahydrofuran (THF) (THF), add 0.857～1.057g Succinic Acid and 0.067～0.08794-Dimethylamino pyridine (DMAP) subsequently, stirring reaction is 2～3 hours at ambient temperature;
Step 5.2: with 30 ℃～40 ℃ concentrating under reduced pressure of reaction soln heating of step 5.1, remove THF, add the dilution of 25～35ml water subsequently, and adopt 25～35ml ethyl acetate extraction 3 times, merge organic phase and adopt saturated common salt water washing, anhydrous sodium sulfate drying, subsequently gained solution is heated to 30 ℃～40 ℃ and is evaporated to driedly, obtain the thick product of the succinate of glaucocalyxin A;
Step 5.3: the thick product of the succinate of glaucocalyxin A is dissolved in the elutriant, and adopts silica gel column chromatography to separate, the gained cut is heated to 30 ℃～40 ℃ concentrating under reduced pressure, remove and desolvate, obtain the clear crystal of the succinate of glaucocalyxin A;
Step 5.4: the succinate of gained glaucocalyxin A is dissolved in 15～20ml tetrahydrofuran (THF) (THF), at room temperature, adds 0.5～0.6g ethylene dichloride (EDC) and 0.2～0.3gN-N-Hydroxysuccinimide (NHS), stirring at room 8～10 hours;
Step 5.5: with the underpressure distillation under 30 ℃～40 ℃ conditions of gained reaction soln, remove THF, adopt 15～20ml washed with dichloromethane subsequently 3 times, use saturated common salt water washing organic phase again, anhydrous sodium sulfate drying filters and with the underpressure distillation under 30 ℃～40 ℃ conditions of gained filtrate, removes and desolvate, dry at ambient temperature, the clear crystal of derivative of the succinate of glaucocalyxin A and NHS;
Step 5.6: at ambient temperature, the glaucocalyxin A derivative of 55～65mg step 5.5 is dissolved in 0.9～1.1mlTHF solution, and with 58～68mg cyclic octapeptide
Be dissolved in 4～6mlPBS buffered soln, the THF drips of solution with the glaucocalyxin A derivative is added in the PBS solution of cyclic octapeptide subsequently, and stirring reaction is 10～20 minutes under the room temperature condition;
Described PBS damping fluid is 0.2 μ mol/L and its pH=8.0;
Step 5.7: THF is removed in reaction soln underpressure distillation under 30 ℃～40 ℃ conditions of step 5.6, subsequent filtration also adopts 1.5～2.5ml water washing 3 times with gained filtrate, merging filtrate, and lyophilize 20～28 hours under-45 ℃～-35 ℃ conditions, the glaucocalyxin A derivative that obtains having the cyclic octapeptide structure.
Embodiment 3:
Adopt following technical parameter to improve embodiment 1.
Adopt in the step 1.1 and be crushed to 30 purpose Herba Rabdosiae glaucocalycis medicinal materials (over-ground part).
In the recrystallization process of step 1.2 and step 1.3, mix gained crushed material and 95% ethanol at 1: 8 according to volume ratio, the temperature of reflux is 83 ℃, and the time of reflux is 1.7 hours.
The recrystallization process of repeating step 1.3 2 times.
In the step 2.1, the temperature of heating concentrating under reduced pressure is 58 ℃.
In the step 2.2, mix elementary enriched material and water at 1: 9 according to volume ratio, the time of leaving standstill after the stirring is 8 hours.
In step 2.3 and 2.4 the recrystallization process, according to the solid and the ethyl acetate of 1: 4.5 mixing step 2.2 gained of volume ratio, the dissolved temperature is 15 ℃, and the time of leaving standstill is 2.5 hours.
The recrystallization process of repeating step 2.4 2 times.
In the step 2.6, the temperature of heating concentrating under reduced pressure is 38 ℃.
In the step 3.1, step 2 gained solid concentrates is dissolved in 95% ethanol according to volume ratio 1: 4.5.
In the step 3.2, adopt the 95% alcohol flushing resin post of 250ml.
In the step 3.3, the temperature of heating reduced-pressure backflow is 58 ℃.
In the step 4.1, mix the polar solvent of residue and 33 ℃ according to volume ratio 1: 4.5, described polar solvent is 1: 1.5 chloroform of volume ratio-acetone mixing solutions, and the temperature of described heating concentrating under reduced pressure is 33 ℃, and the temperature that leaves standstill crystallization is-18 ℃.
In the step 4.2, mix the polar solvent of gained crystal and 33 ℃ according to volume ratio 1: 4.5, described polar solvent is 1: 2 chloroform of volume ratio-acetone mixing solutions, and the temperature of described heating concentrating under reduced pressure is 33 ℃, the temperature that leaves standstill crystallization is-18 ℃, and the time of leaving standstill crystallization is 16 hours.
The recrystallization process of repeating step 4.2 2 times.
Prepare glaucocalyxin A by above-mentioned steps.
Embodiment 4:
Adopt following technical parameter to improve embodiment 1.
Adopt in the step 1.1 and be crushed to 40 purpose Herba Rabdosiae glaucocalycis medicinal materials (over-ground part).
In the recrystallization process of step 1.2 and step 1.3, mix gained crushed material and 95% ethanol at 1: 12 according to volume ratio, the temperature of reflux is 87 ℃, and the time of reflux is 1.3 hours.
The recrystallization process of repeating step 1.3 2 times.
In the step 2.1, the temperature of heating concentrating under reduced pressure is 62 ℃.
In the step 2.2, mix elementary enriched material and water at 1: 9 according to volume ratio, the time of leaving standstill after the stirring is 10 hours.
In step 2.3 and 2.4 the recrystallization process, according to the solid and the ethyl acetate of 1: 5.5 mixing step 2.2 gained of volume ratio, the dissolved temperature is 25 ℃, and the time of leaving standstill is 1.5 hours.
The recrystallization process of repeating step 2.4 2 times.
In the step 2.6, the temperature of heating concentrating under reduced pressure is 42 ℃.
In the step 3.1, step 2 gained solid concentrates is dissolved in 95% ethanol according to volume ratio 1: 5.5.
In the step 3.2, adopt the 95% alcohol flushing resin post of 350ml.
In the step 3.3, the temperature of heating reduced-pressure backflow is 62 ℃.
In the step 4.1, mix the polar solvent of residue and 37 ℃ according to volume ratio 1: 5.5, described polar solvent is 1: 1.5 chloroform of volume ratio-acetone mixing solutions, and the temperature of described heating concentrating under reduced pressure is 37 ℃, and the temperature that leaves standstill crystallization is-18 ℃.
In the step 4.2, mix the polar solvent of gained crystal and 37 ℃ according to volume ratio 1: 5.5, described polar solvent is 1: 3 chloroform of volume ratio-acetone mixing solutions, and the temperature of described heating concentrating under reduced pressure is 37 ℃, the temperature that leaves standstill crystallization is-18 ℃, and the time of leaving standstill crystallization is 20 hours.
The recrystallization process of repeating step 4.2 2 times.
Prepare glaucocalyxin A by above-mentioned steps.
Embodiment 5:
Adopt following technical parameter to improve embodiment 2.
In the step 5.1, each material consumption is glaucocalyxin A 9.26g, THF26ml, Succinic Acid 0.877g, DMAP0.069g.
In the step 5.2, the temperature of concentrating under reduced pressure is 31 ℃, adopts the dilution of 26ml water, adopts the 26ml ethyl acetate extraction, and being evaporated to dried temperature subsequently is 31 ℃.
In the step 5.3, the temperature of gained cut concentrating under reduced pressure is 31 ℃.
In the step 5.4, each material consumption is that THF16ml, EDC0.51g, NHS0.21g stirred 8.5 hours;
In the step 5.5, the temperature of underpressure distillation is 31 ℃, adopts the 16ml washed with dichloromethane, and gained filtrate decompression distillatory temperature is 31 ℃.
In the step 5.6, each material consumption is glaucocalyxin A derivative 56mg, THF0.92ml, cyclic octapeptide 59mg, PBS buffered soln 4.5ml, stirring reaction 11 minutes.
In the step 5.7, vacuum distillation temperature is 31 ℃, adopts the 1.6ml water washing, the lyophilize 21 hours under-44 ℃ of conditions of gained filtrate.
Embodiment 6:
Adopt following technical parameter to improve embodiment 2.
In the step 5.1, each material consumption is glaucocalyxin A 9.66g, THF25～35ml, Succinic Acid 0.917g, DMAP0.073g.
In the step 5.2, the temperature of concentrating under reduced pressure is 33 ℃, adopts the dilution of 28ml water, adopts the 28ml ethyl acetate extraction, and being evaporated to dried temperature subsequently is 33 ℃.
In the step 5.3, the temperature of gained cut concentrating under reduced pressure is 33 ℃.
In the step 5.4, each material consumption is THF17ml, EDC0.53g, NHS0.23g, stirring at room 9 hours;
In the step 5.5, the temperature of underpressure distillation is 33 ℃, adopts the 17ml washed with dichloromethane, and gained filtrate decompression distillatory temperature is 33 ℃.
In the step 5.6, each material consumption is glaucocalyxin A derivative 58mg, THF0.96ml, cyclic octapeptide 61mg, PBS buffered soln 5ml, stirring reaction 13 minutes.
In the step 5.7, vacuum distillation temperature is 33 ℃, adopts the 1.8ml water washing, the lyophilize 23 hours under-42 ℃ of conditions of gained filtrate.
Embodiment 7:
Adopt following technical parameter to improve embodiment 2.
In the step 5.1, each material consumption is glaucocalyxin A 10.06g, THF30ml, Succinic Acid 0.957g, DMAP0.077g.
In the step 5.2, the temperature of concentrating under reduced pressure is 35 ℃, adopts the dilution of 30ml water, adopts the 30ml ethyl acetate extraction, and being evaporated to dried temperature subsequently is 35 ℃.
In the step 5.3, the temperature of gained cut concentrating under reduced pressure is 35 ℃.
In the step 5.4, each material consumption is THF17.5ml, EDC0.55g, NHS0.25g, stirring at room 9 hours;
In the step 5.5, the temperature of underpressure distillation is 35 ℃, adopts the 17.5ml washed with dichloromethane, and gained filtrate decompression distillatory temperature is 35 ℃.
In the step 5.6, each material consumption is glaucocalyxin A derivative 60mg, THF1.00ml, cyclic octapeptide 63mg, PBS buffered soln 5ml, stirring reaction 15 minutes.
In the step 5.7, vacuum distillation temperature is 35 ℃, adopts the 2.0ml water washing, the lyophilize 24 hours under-40 ℃ of conditions of gained filtrate.
Embodiment 8:
Adopt following technical parameter to improve embodiment 2.
In the step 5.1, each material consumption is glaucocalyxin A 10.46g, THF25～35ml, Succinic Acid 0.997g, DMAP0.081g.
In the step 5.2, the temperature of concentrating under reduced pressure is 37 ℃, adopts the dilution of 32ml water, adopts the 32ml ethyl acetate extraction, and being evaporated to dried temperature subsequently is 37 ℃.
In the step 5.3, the temperature of gained cut concentrating under reduced pressure is 37 ℃.
In the step 5.4, each material consumption is THF18ml, EDC0.57g, NHS0.27g, stirring at room 9 hours;
In the step 5.5, the temperature of underpressure distillation is 37 ℃, adopts the 18ml washed with dichloromethane, and gained filtrate decompression distillatory temperature is 37 ℃.
In the step 5.6, each material consumption is glaucocalyxin A derivative 62mg, THF1.04ml, cyclic octapeptide 65mg, PBS buffered soln 5ml, stirring reaction 17 minutes.
In the step 5.7, vacuum distillation temperature is 37 ℃, adopts the 2.2ml water washing, the lyophilize 25 hours under-38 ℃ of conditions of gained filtrate.
Embodiment 9:
Adopt following technical parameter to improve embodiment 2.
In the step 5.1, each material consumption is glaucocalyxin A 10.86g, THF25～35ml, Succinic Acid 1.037g, DMAP0.083g.
In the step 5.2, the temperature of concentrating under reduced pressure is 39 ℃, adopts the dilution of 34ml water, adopts the 34ml ethyl acetate extraction, and being evaporated to dried temperature subsequently is 39 ℃.
In the step 5.3, the temperature of gained cut concentrating under reduced pressure is 39 ℃.
In the step 5.4, each material consumption is THF19ml, EDC0.59g, NHS0.29g, stirring at room 9.5 hours;
In the step 5.5, the temperature of underpressure distillation is 39 ℃, adopts the 19ml washed with dichloromethane, and gained filtrate decompression distillatory temperature is 39 ℃.
In the step 5.6, each material consumption is glaucocalyxin A derivative 64mg, THF1.08ml, cyclic octapeptide 67mg, PBS buffered soln 5.5ml, stirring reaction 19 minutes.
In the step 5.7, vacuum distillation temperature is 39 ℃, adopts the 2.4ml water washing, the lyophilize 27 hours under-36 ℃ of conditions of gained filtrate.
Embodiment 10:
Technical parameter is respectively with embodiment 7～9, and just among each embodiment, the cyclic octapeptide that is adopted is a D type cyclic octapeptide
Embodiment 11:
Technical parameter is respectively with embodiment 7～9, and just among each embodiment, the cyclic octapeptide that is adopted is a L type cyclic octapeptide
Embodiment 12:
Prepare glaucocalyxin A according to embodiment 3～4, and the gained glaucocalyxin A is carried out derivatize according to embodiment 5～9,, prepare glaucocalyxin A derivative with D type cyclic octapeptide structure particularly according to embodiment 10.
Adopt high performance liquid chromatography (HPLC) and the above-mentioned glaucocalyxin A derivant structure of mass spectrometric detection, gained HPLC spectrogram and mass spectrum are shown in Fig. 3 a and 3b.Show that through HPLC and mass spectrometric detection above-mentioned glaucocalyxin A derivant structure is as follows.
Embodiment 13:
Prepare glaucocalyxin A according to embodiment 3～4, and the gained glaucocalyxin A is carried out derivatize according to embodiment 5～9,, prepare glaucocalyxin A derivative with L type cyclic octapeptide structure particularly according to embodiment 11.
Adopt high performance liquid chromatography (HPLC) and the above-mentioned glaucocalyxin A derivant structure of mass spectrometric detection, gained HPLC spectrogram and mass spectrum are shown in Fig. 5 a and 5b.Show that through HPLC and mass spectrometric detection above-mentioned glaucocalyxin A derivant structure is as follows.
Embodiment 14: the preparation with glaucocalyxin A derivative formulations of cyclic octapeptide structure
Prepare the glaucocalyxin A derivative with cyclic octapeptide structure of structural formula with embodiment 12 and 13 according to the foregoing description.
The glaucocalyxin A derivative that gained is had the cyclic octapeptide structure, adopt the conventional medicine carrier on the pharmaceutics, and the conventional preparation method on the employing pharmaceutics, be prepared into the regular dosage form on the pharmaceutics, include but not limited to tablet, capsule, flexible glue agent, sprays, gelifying agent, gel inhalation, oral preparation, suspensoid, electuary, patch, ointment, pill, powder, injection, infusion solution, freeze dried injection, lipidosome injection, target administration injection, suppository, sustained release preparation or controlled release preparation.
Embodiment 15: the glaucocalyxin A derivative with cyclic octapeptide structure is at the SMMC-7721 human liver cancer cell, the HepG2 human liver cancer cell, SW-1990 human pancreas cholangiocarcinoma cell, the HO-8910 Proliferation of Human Ovarian Cell, the K-562 tumour cell, the KB tumour cell, MCF-7 breast cancer tumour cell, Eca-109 human esophagus cancer tumour cell, A-549 lung cancer tumour cell, kidney A-498 tumour cell, cervical cancer HeLa tumour cell, the inhibiting pharmacodynamic experiment of human colon carcinoma SW480 growth of tumour cell
1, medicine and reagent:
Given the test agent, F-12 substratum, 10% inactivated fetal bovine serum (FBS), dimethyl sulfoxide (DMSO) (DMSO), thiazole salt (MTT), Zorubicin (positive drug)
2, instrument:
Incubator, super quiet worktable, multi-functional inversion microscope, whizzer, automatic microplate reader, 96 well culture plates
3, cell strain:
SMMC-7721 human liver cancer cell, HepG2 human liver cancer cell, SW-1990 human pancreas cholangiocarcinoma, HO-8910 Proliferation of Human Ovarian Cell, K-562 tumour cell, KB tumour cell, MCF-7 tumour cell, Eca-109 human esophagus cancer tumour cell, A-549 lung cancer tumour cell, kidney A-498 tumour cell, cervical cancer HeLa tumour cell, human colon carcinoma SW480 tumour cell
4, sample preparation:
Get according to what the foregoing description prepared and have the substituent glaucocalyxin A derivative of D type cyclic octapeptide, have the substituent glaucocalyxin A derivative of L type cyclic octapeptide, dimethyl sulfoxide (DMSO) (DMSO) dissolving that adds respective volume respectively, obtain the drug solution of concentration for about 50mg/ml, concussion dissolving subsequently, the gained drug solution can store under-20 ℃ of conditions
5, experimental technique:
With above-mentioned tumor cell culture in the F-12 substratum that contains 10%FBS.The above-mentioned tumour cell of taking the logarithm vegetative period carries out cell counting after the digestion routinely.Adjusting cell concn after the cell counting is 1 * 10
4And be inoculated on the 96 porocyte culture plates, place incubator to cultivate dosing after 24 hours.Control group changes nutrient solution after 24 hours, and experimental group adds the thing that tried of different concns, uses mtt assay (MTT colorimetry, thiazole blue laws) colorimetric detection cell viability after 48 hours.Adding concentration in every hole is MTT (tetrazolium bromide) the 20 μ l of 5mg/ml, cultivates to inhale after 4 hours and removes nutrient solution, adds DMSO150 μ l, and the shaking table vibration is dissolved the bluish voilet deposit seeds fully, and the 490nm place surveys absorbancy (A) value on microplate reader.
6, experimental result:
Glaucocalyxin A derivative with cyclic octapeptide structure is as shown in the table to the influence of SMMC-7721 human liver cancer cell, HepG2 human liver cancer cell, SW-1990 human pancreas cholangiocarcinoma, HO-8910 Proliferation of Human Ovarian Cell, K-562 tumour cell, KB tumour cell, MCF-7 tumour cell, Eca-109 human esophagus cancer tumour cell, A-549 lung cancer tumour cell, kidney A-498 tumour cell, cervical cancer HeLa tumour cell, human colon carcinoma SW480 tumor cell proliferation
Annotate: F1 represents to have the substituent glaucocalyxin A derivative of D type cyclic octapeptide;
F2 represents to have the substituent glaucocalyxin A derivative of L type cyclic octapeptide.
The result shows, glaucocalyxin A derivative with cyclic octapeptide structure can obviously suppress the growth of SMMC-7721 human liver cancer cell, HepG2 human liver cancer cell, SW-1990 human pancreas cholangiocarcinoma, HO-8910 Proliferation of Human Ovarian Cell, K-562 tumour cell, KB tumour cell, MCF-7 tumour cell, Eca-109 human esophagus cancer tumour cell, A-549 lung cancer tumour cell, kidney A-498 tumour cell, cervical cancer HeLa tumour cell, human colon carcinoma SW480 tumour cell, and this effect is irrelevant with solubility promoter DMSO.
Embodiment 16: the glaucocalyxin A derivative with cyclic octapeptide structure is at cancer of the stomach ags cell Growth Inhibition pharmacodynamic experiment
1, medicine and reagent:
Given the test agent, F-12 substratum, 10% inactivated fetal bovine serum (FBS), dimethyl sulfoxide (DMSO) (DMSO), thiazole salt (MTT), Zorubicin (positive drug)
2, instrument:
Incubator, super quiet worktable, multi-functional inversion microscope, whizzer, automatic microplate reader, 96 well culture plates
3, cell strain:
The cancer of the stomach ags cell
4, sample preparation:
Get according to what the foregoing description prepared and have the substituent glaucocalyxin A derivative of D type cyclic octapeptide, have the substituent glaucocalyxin A derivative of L type cyclic octapeptide, dimethyl sulfoxide (DMSO) (DMSO) dissolving that adds respective volume respectively, obtain the drug solution of concentration for about 50mg/ml, concussion dissolving subsequently, the gained drug solution can store under-20 ℃ of conditions
5, experimental technique:
With above-mentioned tumor cell culture in the F-12 substratum that contains 10%FBS.The above-mentioned tumour cell of taking the logarithm vegetative period carries out cell counting after the digestion routinely.Adjusting cell concn after the cell counting is 1 * 10
4And be inoculated on the 96 porocyte culture plates, place incubator to cultivate dosing after 24 hours.Control group changes nutrient solution after 24 hours, and experimental group adds the thing that tried of different concns, uses mtt assay (MTT colorimetry, thiazole blue laws) colorimetric detection cell viability after 48 hours.Adding concentration in every hole is MTT (tetrazolium bromide) the 20 μ l of 5mg/ml, cultivates to inhale after 4 hours and removes nutrient solution, adds DMSO 150 μ l, and the shaking table vibration is dissolved the bluish voilet deposit seeds fully, and the 490nm place surveys absorbancy (A) value on microplate reader.
6, experimental result:
Glaucocalyxin A derivative with cyclic octapeptide structure is as shown in the table to the influence of cancer of the stomach ags cell propagation
| Group | Concentration | A value (490nm) | Inhibiting rate (%) |
| Blank | - | 0.4420+0.0355 | |
| F1 | 1μmol/ml | 0.0121+0.0182 ＊＊△△ | 94.11 |
| 0.1μmol/ml | 0.0293+0.0190 ＊＊△△ | 89.88 | |
| 0.01μmol/ml | 0.0590+0.0251 ＊＊△△ | 78.23 | |
| F2 | 1μmol/ml | 0.0280+0.0188 ＊＊△△ | 92.41 |
| 0.1μmol/ml | 0.0402+0.0177 ＊＊△△ | 81.45 | |
| 0.01μmol/ml | 0.0651+0.0204 ＊＊△△ | 76.44 | |
| The DMSO contrast | 1μl/ml | 0.3723+0.0492 | 15.76 |
| 0.1μl/ml | 0.4417+0.0318 | 0.08 | |
| 0.01l/ml | 0.4070+0.0288 | 7.92 |
Annotate:
*Expression is compared P＜0.05 with the blank group;
*Expression is compared P＜0.01 with the blank group;
△ represents to compare P＜0.05 with the DMSO control group of same concentrations;
△ △ represents to compare P＜0.01 with the DMSO control group of same concentrations;
F1 represents to have the substituent glaucocalyxin A derivative of D type cyclic octapeptide;
F2 represents to have the substituent glaucocalyxin A derivative of L type cyclic octapeptide.
The result shows that the glaucocalyxin A derivative with cyclic octapeptide structure can obviously suppress the growth of cancer of the stomach ags cell, and this effect is irrelevant with solubility promoter DMSO.
Embodiment 17: have the influence of the glaucocalyxin A derivative of cyclic octapeptide structure to hormonal dependent human prostata cancer LNCap cell proliferation
1, medicine and reagent:
Given the test agent, F-12 substratum, 10% inactivated fetal bovine serum (FBS), dimethyl sulfoxide (DMSO) (DMSO), thiazole salt (MTT), Zorubicin (positive drug)
2, instrument:
Incubator, super quiet worktable, multi-functional inversion microscope, whizzer, automatic microplate reader, 96 well culture plates
3, cell strain:
The cancer of the stomach ags cell
4, sample preparation:
Get according to what the foregoing description prepared and have the substituent glaucocalyxin A derivative of D type cyclic octapeptide, have the substituent glaucocalyxin A derivative of L type cyclic octapeptide, dimethyl sulfoxide (DMSO) (DMSO) dissolving that adds respective volume respectively, obtain the drug solution of concentration for about 50mg/ml, concussion dissolving subsequently, the gained drug solution can store under-20 ℃ of conditions
5, experimental technique:
Recovery LNCaP cell cultures is in the F-12 substratum that contains 10%FBS.Grow after 2-3 days, put biomicroscope observation of cell adherent growth situation.When cell grow to be paved with bottle at the bottom of the time, the original fluid in the disposable Celllbind culturing bottle that exhausts adds 0.25% trypsinase that contains EDTA and digests, and culturing bottle is set level, and builds bottle cap, mirror is observed the digestion situation down.After digesting about 1～2 minute, obviously retraction appears in observation of cell under the mirror, and the intercellular substance change is big, when breaking away from the bottle wall, adds and contains 10%FBS F-12 substratum termination digestion.Liquid is absorbed in the 15ml centrifuge tube centrifugal 5 minutes of 1500rpm.Take out centrifuge tube, inhale and abandon supernatant, add 10%FBS F-12 substratum 1ml again, after the suitable power piping and druming evenly, adjusting cell concn after the cell counting is 1 * 10
5Be inoculated on the 96 porocyte culture plates, place incubator to cultivate dosing after 48 hours.Control group changes nutrient solution after 48 hours, and experimental group adds the thing that tried of different concns, uses mtt assay colorimetric detection cell viability after 24 hours.MTT (tetrazolium bromide) the 20 μ l that add 5mg/ml in every hole cultivate to inhale after 4 hours and remove nutrient solution, add DMSO150 μ l, and the shaking table vibration is dissolved the bluish voilet deposit seeds fully, and the 490nm place surveys absorbancy (A) value on microplate reader.
5, positive control
Get a slice ratio karoo amine slice (about 50mg), add distilled water 120ml, obtaining concentration is the solution of 0.42mg/ml (1000 μ mol/L), and with the filtering with microporous membrane of 0.22 μ m ,-20 ℃ of preservations are standby.
6, experimental result:
Glaucocalyxin A derivative with cyclic octapeptide structure is as shown in the table to the influence of hormonal dependent human prostata cancer LNCap cell proliferation
| Group | Concentration | A value (490nm) | Inhibiting rate (%) |
| Blank | - | 1.4133±0.0433 | |
| F1 | 1μmol/ml | 0.0223+0.0182 ＊＊△△ | 91.36 |
| 0.1μmol/ml | 0.0341+0.0190 ＊＊△△ | 85.98 | |
| 0.01μmol/ml | 0.0513+0.0251 ＊＊△△ | 79.72 | |
| F2 | 1μmol/ml | 0.0042±0.0047 ＊＊△△ | 99.71 |
| 0.1μmol/ml | 0.1524±0.0576 ＊＊△△ | 89.22 | |
| 0.01μmol/ml | 1.5590+-.0490 ＊ | -10.31 | |
| The DMSO contrast | 1μl/ml | 1.2262+0.0178 ＊＊ | 13.24 |
| 0.1μl/ml | 1.2785+0.2089 | 9.54 | |
| 0.01l/ml | 1.6083+0.0861 ＊ | -13.79 |
Annotate:
*Expression is compared P＜0.05 with the blank group;
*Expression is compared P＜0.01 with the blank group;
△ represents to compare P＜0.05 with the DMSO control group of same concentrations;
△ △ represents to compare P＜0.01 with the DMSO control group of same concentrations;
F1 represents to have the substituent glaucocalyxin A derivative of D type cyclic octapeptide;
F2 represents to have the substituent glaucocalyxin A derivative of L type cyclic octapeptide.
The result shows that the glaucocalyxin A derivative with cyclic octapeptide structure can obviously suppress the propagation of people's hormonal dependent prostate cancer LNCaP cell, and this effect is irrelevant with solubility promoter DMSO.
Embodiment 18: have the growth-inhibiting effect of the glaucocalyxin A derivative of cyclic octapeptide structure to the HL-60 tumor cell line
1, medicine and reagent:
Given the test agent, F-12 substratum, 10% inactivated fetal bovine serum (FBS), dimethyl sulfoxide (DMSO) (DMSO), thiazole salt (MTT), Zorubicin (positive drug)
2, instrument:
Incubator, super quiet worktable, multi-functional inversion microscope, whizzer, automatic microplate reader, 96 well culture plates
3, cell strain:
The HL-60 tumour cell
4, sample preparation:
Get according to what the foregoing description prepared and have the substituent glaucocalyxin A derivative of D type cyclic octapeptide, have the substituent glaucocalyxin A derivative of L type cyclic octapeptide, dimethyl sulfoxide (DMSO) (DMSO) dissolving that adds respective volume respectively, obtain the drug solution of concentration for about 50mg/ml, concussion dissolving subsequently, the gained drug solution can store under-20 ℃ of conditions
5, experimental technique:
With above-mentioned tumor cell culture in the F-12 substratum that contains 10%FBS.The above-mentioned tumour cell of taking the logarithm vegetative period carries out cell counting after the digestion routinely.Adjusting cell concn after the cell counting is 1 * 10
4And be inoculated on the 96 porocyte culture plates, place incubator to cultivate dosing after 24 hours.Control group changes nutrient solution after 24 hours, and experimental group adds the thing that tried of different concns, uses mtt assay (MTT colorimetry, thiazole blue laws) colorimetric detection cell viability after 48 hours.Adding concentration in every hole is MTT (tetrazolium bromide) the 20 μ l of 5mg/ml, cultivates to inhale after 4 hours and removes nutrient solution, adds DMSO150 μ l, and the shaking table vibration is dissolved the bluish voilet deposit seeds fully, and the 490nm place surveys absorbancy (A) value on microplate reader.
5, experimental result:
Glaucocalyxin A derivative with cyclic octapeptide structure is as shown in the table to HL-60 growth of tumour cell restraining effect
| Group | Concentration | A value (490nm) | Inhibiting rate (%) |
| Blank | - | 1.4206±0.0419 | |
| A3 | 1μmol/ml | 0.0187+0.0182 ＊＊△△ | 94.38 |
| 0.1μmol/ml | 0.0417+0.0190 ＊＊△△ | 76.42 | |
| 0.01μmol/ml | 1.1609+0.0251 ＊＊△△ | 18.66 | |
| A4 | 1μmol/ml | 0.0151±0.0039 ＊＊△△ | 95.32 |
| 0.1μmol/ml | 0.0478±0.0412 ＊＊△△ | 72.44 | |
| 0.01μmol/ml | 1.3904+-0.460 ＊ | -8.49 | |
| The DMSO contrast | 1μl/ml | 1.3098+0.0178 ＊＊ | 10.24 |
| 0.1μl/ml | 1.3241+0.2089 | 9.54 | |
| 0.01l/ml | 1.6083+0.0861 ＊ | -13.79 |
Annotate:
*Expression is compared P＜0.05 with the blank group;
*Expression is compared P＜0.01 with the blank group;
△ represents to compare P＜0.05 with the DMSO control group of same concentrations;
△ △ represents to compare P＜0.01 with the DMSO control group of same concentrations;
F1 represents to have the substituent glaucocalyxin A derivative of D type cyclic octapeptide;
F2 represents to have the substituent glaucocalyxin A derivative of L type cyclic octapeptide.
The result shows that the glaucocalyxin A derivative with cyclic octapeptide structure can obviously suppress the propagation of HL-60 tumour cell, and this effect is irrelevant with solubility promoter DMSO.
Embodiment 19: the application with glaucocalyxin A derivative formulations of cyclic octapeptide structure
According to embodiment 15～18, prepare the glaucocalyxin A derivative with cyclic octapeptide structure of structural formula with embodiment 12～13 according to the foregoing description, be used for treatment for cancer, particularly the application in treatment cancer of the stomach, prostate gland, leukemia, nasopharyngeal carcinoma, liver cancer, pancreas cholangiocarcinoma, ovarian cancer, mammary cancer, the esophageal carcinoma, lung cancer, kidney, cervical cancer, colorectal carcinoma.
Foregoing is exemplifying of specific embodiments of the invention, for the wherein not reagent of detailed description, equipment, working method etc., should be understood to take the existing common and conventional reagent in this area, equipment, working method to wait and implemented.
The above embodiment of the present invention for the usefulness of explanation technical solution of the present invention, is enumerating of technical solution of the present invention only only simultaneously, is not limited to technical scheme of the present invention and protection domain thereof.Adopt the equivalent technologies means, be equal to reagent etc. the improvement of claims of the present invention and the disclosed technical scheme of specification sheets be will be understood that it is not exceed claims of the present invention and the disclosed scope of specification sheets.
Claims (13)
1. the glaucocalyxin A derivative with cyclic octapeptide structure is characterized in that, is a kind of kaurane type tetracyclic diterpene class material, has kaurane type structure, carbonyl, hydroxyl and cyclic octapeptide structure, and the general formula of its molecular structure is as follows:
Wherein, R1, R2 be-OH or
Wherein, R3 is the cyclic octapeptide substituting group.
2. the glaucocalyxin A derivative with cyclic octapeptide structure as claimed in claim 1 is characterized in that, has one to be-OH among described R1 and the R2.
3. polypeptide class glaucocalyxin A derivative as claimed in claim 1 is characterized in that described R3 is a cyclic octapeptide
The molecular formula of described cyclic octapeptide is C33H48N11O17S2.
4. the glaucocalyxin A derivative with cyclic octapeptide structure as claimed in claim 3 is characterized in that, described R3 is a D type cyclic octapeptide substituting group
Described R1 is
Described R2 is-OH that its molecular structure is as follows:
5. the glaucocalyxin A derivative with cyclic octapeptide structure as claimed in claim 3 is characterized in that, described R3 is a D type cyclic octapeptide substituting group
Described R2 is
Described R1 is-OH that its molecular structure is as follows:
6. the glaucocalyxin A derivative with cyclic octapeptide structure as claimed in claim 3 is characterized in that, described R3 is a L type cyclic octapeptide substituting group
Described R1 is
Described R2 is-OH that its molecular structure is as follows:
7. the glaucocalyxin A derivative with cyclic octapeptide structure as claimed in claim 3 is characterized in that, described R3 is a L type cyclic octapeptide substituting group
Described R2 is
Described R1 is-OH that its molecular structure is as follows:
8. the preparation method with glaucocalyxin A derivative of cyclic octapeptide structure as claimed in claim 1 is characterized in that, comprises following steps:
Step 1: extract
Step 1.1: get Herba Rabdosiae glaucocalycis medicinal material (over-ground part) and be crushed to 20 orders～50 orders;
Step 1.2: the crushed material of gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 5～1: 15, and heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, and obtained extracting solution and residuum;
Step 1.3: the residuum of step 1.2 gained was mixed according to volume ratio with 95% ethanol (A.R.) in 1: 5～1: 15, heating refluxed 1～2 hour under 80 ℃～90 ℃ temperature condition, extracted, filtered, obtain extracting solution and residuum, repeat this step 1～3 time;
Step 1.4: the extracting solution of combining step 1.2 and 1.3 gained;
Step 2: solvent treatment
Step 2.1: with the extracting solution heating of step 1 gained, under 55 ℃～65 ℃ temperature condition, heat concentrating under reduced pressure, reclaim ethanol and obtain the elementary enriched material of heavy-gravity;
Step 2.2: the elementary enriched material of step 2.1 gained was mixed according to volume ratio with water in 1: 8～1: 10, stirred 10 minutes with 60～120 rev/mins speed at ambient temperature, left standstill subsequently 6～12 hours, abandoning supernatant obtains lower floor's solid;
Step 2.3: the solid and the ethyl acetate (A.R.) of step 2.2 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition,, left standstill 1～3 hour, filter and obtain filtrate and insolubles with 60～120 rev/mins speed stirring and dissolving;
Step 2.4: the insolubles and the ethyl acetate (A.R.) of step 2.3 gained were mixed according to volume ratio in 1: 4～1: 6, under 25 ℃～35 ℃ temperature condition with 60～120 rev/mins speed stirring and dissolving, left standstill 1～3 hour, and filtered and to obtain filtrate and insolubles, repeat this step 1～3 time;
Step 2.5: the filtrate of combining step 2.3 and 2.4 gained;
Step 2.6: with the filtrate heating of step 2.5 gained, under 35 ℃～45 ℃ temperature condition, heat concentrating under reduced pressure, reclaim ethyl acetate (A.R.), obtain solid concentrates;
Step 3: plastic resin treatment
Step 3.1: the solid concentrates of step 2 gained and 95% ethanol (A.R.) according to 1: 4～1: 6 mixed dissolution of volume ratio, are obtained solution;
Step 3.2: the solution of step 3.1 gained slowly is added in No. 7 resin columns, work the color of observing resin, when resin column has 2/3 variable color, stop application of sample, and adopt 95% ethanol (A.R.) of 200ml～400ml to wash this resin column, collect all effluent liquid, the weighting material of described resin column is a strongly basic anionic resin and to be saturated to the pH value with sodium hydroxide neutral;
Step 3.3: with the heating of step 3.2 gained effluent liquid, and under 55 ℃～65 ℃ temperature condition, heat reduced-pressure backflow, do, obtain solid residue until solvent;
Step 4: recrystallization
Step 4.1: the polar solvent of above-mentioned residue with 30 ℃～40 ℃ mixed according to volume ratio in 1: 4～1: 6, obtain primary solution, heating, concentrating under reduced pressure under 30 ℃～40 ℃ temperature condition, under-23 ℃～-13 ℃ temperature condition, leave standstill crystallization, filter and obtain light yellow needle crystal;
Described polar solvent is the mixing solutions according to 1: 1～1: 2 blended chloroform (A.R.) of volume ratio and acetone (A.R.);
Step 4.2: the crystallization of step 4.1 gained was mixed according to volume ratio with polar solvent in 1: 4～1: 6, and recrystallization 2～4 times repeatedly under-23 ℃～-13 ℃ temperature condition each 12～24 hours, obtains the intermediate product glaucocalyxin A;
Described polar solvent is the mixing solutions according to 1: 1～1: 4 blended chloroform (A.R.) of volume ratio and acetone (A.R.);
Step 5: derivatize
At room temperature, step 4 gained is dissolved in glaucocalyxin A in the tetrahydrofuran (THF) (THF), add Succinic Acid and 4-Dimethylamino pyridine (DMAP) subsequently, stirring reaction, pressure reducing and steaming tetrahydrofuran (THF) subsequently, extract the succinate of glaucocalyxin A, subsequently it is obtained as the succinate of the glaucocalyxin A of intermediate product and the derivative of NHS with N-hydroxy-succinamide (NHS) reaction under ethylene dichloride (EDC) condition, subsequently it is dissolved in THF solution, and cyclic octapeptide is dissolved in phosphate buffer soln (PBS buffered soln), subsequently at ambient temperature, the THF drips of solution of glaucocalyxin A derivative is added in the PBS solution of cyclic octapeptide, stirring reaction, THF is removed in underpressure distillation subsequently, gained filtrate obtains having the glaucocalyxin A derivative of cyclic octapeptide structure after lyophilize.
9. the preparation method with glaucocalyxin A derivative of cyclic octapeptide structure as claimed in claim 8 is characterized in that described cyclic octapeptide is
10. the preparation method with glaucocalyxin A derivative of cyclic octapeptide structure as claimed in claim 8 is characterized in that described step 5 further may further comprise the steps:
Step 5.1: at room temperature, 9.06～11.06g step 4 gained is dissolved in glaucocalyxin A in 25～35ml tetrahydrofuran (THF) (THF), add 0.857～1.057g Succinic Acid and 0.067～0.087g4-Dimethylamino pyridine (DMAP) subsequently, stirring reaction is 2～3 hours at ambient temperature;
Step 5.2: with 30 ℃～40 ℃ concentrating under reduced pressure of reaction soln heating of step 5.1, remove THF, add the dilution of 25～35ml water subsequently, and adopt 25～35ml ethyl acetate extraction 3 times, merge organic phase and adopt saturated common salt water washing, anhydrous sodium sulfate drying, subsequently gained solution is heated to 30 ℃～40 ℃ and is evaporated to driedly, obtain the thick product of the succinate of glaucocalyxin A;
Step 5.3: the thick product of the succinate of glaucocalyxin A is dissolved in the elutriant, and adopts silica gel column chromatography to separate, the gained cut is heated to 30 ℃～40 ℃ concentrating under reduced pressure, remove and desolvate, obtain the clear crystal of the succinate of glaucocalyxin A;
Step 5.4: the succinate of gained glaucocalyxin A is dissolved in 15～20ml tetrahydrofuran (THF) (THF), at room temperature, adds 0.5～0.6g ethylene dichloride (EDC) and 0.2～0.3gN-N-Hydroxysuccinimide (NHS), stirring at room 8～10 hours;
Step 5.5: with the underpressure distillation under 30 ℃～40 ℃ conditions of gained reaction soln, remove THF, adopt 15～20ml washed with dichloromethane subsequently 3 times, use saturated common salt water washing organic phase again, anhydrous sodium sulfate drying filters and with the underpressure distillation under 30 ℃～40 ℃ conditions of gained filtrate, removes and desolvate, dry at ambient temperature, the clear crystal of derivative of the succinate of glaucocalyxin A and NHS;
Step 5.6: at ambient temperature, the glaucocalyxin A derivative of 55～65mg step 5.5 is dissolved in 0.9～1.1mlTHF solution, and with 58～68mg cyclic octapeptide
Be dissolved in 4～6mlPBS buffered soln, the THF drips of solution with the glaucocalyxin A derivative is added in the PBS solution of cyclic octapeptide subsequently, and stirring reaction is 10～20 minutes under the room temperature condition;
Step 5.7: THF is removed in reaction soln underpressure distillation under 30 ℃～40 ℃ conditions of step 5.6, subsequent filtration also adopts 1.5～2.5ml water washing 3 times with gained filtrate, merging filtrate, and lyophilize 20～28 hours under-45 ℃～-35 ℃ conditions, the glaucocalyxin A derivative that obtains having the cyclic octapeptide structure.
11. the preparation method with glaucocalyxin A derivative of cyclic octapeptide structure as claimed in claim 8 is characterized in that described cyclic octapeptide
Be the D type.
12. the preparation method with glaucocalyxin A derivative of cyclic octapeptide structure as claimed in claim 8 is characterized in that described cyclic octapeptide
Be the L type.
13. as the described application of glaucocalyxin A derivative in the treatment cancer, the particularly application in treatment cancer of the stomach, prostate gland, leukemia, nasopharyngeal carcinoma, liver cancer, pancreas cholangiocarcinoma, ovarian cancer, mammary cancer, the esophageal carcinoma, lung cancer, kidney, cervical cancer, colorectal carcinoma of claim 1～7 with cyclic octapeptide structure.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106631804A (en) * | 2016-12-21 | 2017-05-10 | 江苏省中医药研究院 | Compound separated out from labiatae isodon plant, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106631804A (en) * | 2016-12-21 | 2017-05-10 | 江苏省中医药研究院 | Compound separated out from labiatae isodon plant, and preparation method and application thereof |
| CN106631804B (en) * | 2016-12-21 | 2019-02-15 | 江苏省中医药研究院 | A kind of compound and the preparation method and application thereof isolated from Labiatae Rabdosia plant |
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