CN1706845A - Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid - Google Patents
Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid Download PDFInfo
- Publication number
- CN1706845A CN1706845A CN 200410045600 CN200410045600A CN1706845A CN 1706845 A CN1706845 A CN 1706845A CN 200410045600 CN200410045600 CN 200410045600 CN 200410045600 A CN200410045600 A CN 200410045600A CN 1706845 A CN1706845 A CN 1706845A
- Authority
- CN
- China
- Prior art keywords
- alkaloid
- reaction product
- krs
- alkylating agent
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention prepares alkaloid reacting product through the following steps: the reaction of alkaloid to alkylating agent and subsequent washing with water or proper solvent to eliminate un-reacted alkylating agent and other water soluble components from the reacted mixture; and treating the reacted mixture with strong acid, preferably HCl, to deposit water soluble salt in the reacted product. The precipitate includes at least one kind of quaternary ammonium alkaloid derivative capable of being applied as medicine for preventing and treating diseases, especially immune disorder, metabolic disorder and cancer.
Description
Invention field
The present invention has related generally to drug development and sanitation and health-care field, and it is relevant with pilewort alkaloid and derivative thereof, and wherein, the nitrogen in the pilewort alkaloid molecule is quaternary nitrogen.In addition, the present invention also with the making method of this compound, comprise the mixture of this compound and relevant to the treatment of various diseases and physical appearance.
State-of-art
Pilewort alkaloid and to contain the alkaloidal mixture of pilewort well-known in this technical field is used because they are pilewort alkaloid or the treatment of some pilewort alcaloid-derivatives aspect various physical appearances of treatment and disease (comprising metabolism disorder and tumour).
By some Chelidonium majus L. alkaloid and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide is reacted the preparation that DE 2 028 330 and US 3 865 830 have disclosed thio-phosphamide-isoquinoline 99.9 adducts in organic solvent.
By reacting with the phosphorus compound of system cancer (salt by converting it into them and provide with water-soluble form), AT354 644 and AT 377 988 have described alkaloidal phosphorus derivant preparation technology.This openly shortcoming of technology is that reaction product does not change into water-soluble salt fully, and most of reaction product is still water insoluble.
US 5 981 512 has disclosed the effectiveness of material when the treatment radiation injury that is drawn in AT 377 988 and AT 354 644.
The compound of describing in the above-mentioned patent has different supression cell growths and system cancer activity.Alkaloid mixture, the especially total alkaloids of Chelidonium majus L. have proved to promise to be cancer curing medicine most, and imitating approximately wherein showed in the multinomial research of relevant cancer therapy.Remove the synthetic mixture of unreacted reactant after reaction is finished.(below be also referred to as " thiotepa ") dissolves in the organic solvents such as benzene, ether or chloroform because three (1-acridyl) phosphuret-(t)ed hydrogen sulfide, in the suggestion processing method formerly, unreacted three (1-acridyl) phosphuret-(t)ed hydrogen sulfide is removed from the synthetic mixture with ether washing reaction product.
Even need utilize the inflammable volatile organic solvent purification final product that has equally although above-mentioned manufacturing has the previous processing method of the pilewort alcaloid-derivatives of pharmaceutical activity, but find at present, can also use water solvent to finish purification, like this even can obtain better effect.
The present invention's summary
On the one hand, the novel preparation process that the present invention has related to the alkaloid reaction product of utilizing corresponding alkylating agent to carry out---especially pilewort alkaloid, Oxychelidonine or methoxyl group pilewort alkaloid---, this technology is minimum to comprise the step that a step utilizes water solvent (preferably water) to wash after alkylation reaction is finished.
This technology has comprised also that alcaloid-derivatives is changed into water-soluble salt is rapid, to make hypotoxicity and to have the step of the injectable medicament of wide action spectrum scope.
On the other hand, the present invention relates to water soluble reaction products, for example synthetic pilewort alcaloid-derivatives, wherein the binary nitrogen of this alkaloid molecule beginning has been converted to quaternary nitrogen, wherein the 4th of this quaternary nitrogen the dentate seldom is the alkyl atomic group, and be methyl or ethyl atomic group mostly, or alternate methyl or ethyl atomic group, for example thiotepa atomic group.Optionally gather during in concrete form at first, the character that quaternary amine pilewort alcaloid-derivatives has is as at destination organization---especially cancerous tissue---.
On the other hand, the present invention relates to comprise above-mentioned wherein a kind of quaternary amine alkaloidal pharmaceutical cpd, especially quaternary amine pilewort alkaloid---can be from according to the derivative that obtains the technology of the present invention.
The invention still further relates to the reaction product that comprises as the quaternary amine alcaloid-derivatives of medical medicine, and the pharmaceutical cpd that said derivative is used to make medical various diseases or physical appearance.
The others that the present invention relates to are illustrated in following requirement.
The present invention describes in detail
The process of carrying out according to the present invention has comprised having alkylating agent and (preferably itself has had medical active alkylating agent, for example cytotoxinic phosphamide or comprise the phosphoric acid derivatives of an acridyl at least) organic solvent in alkaloid or alkaloid mixture are reacted, wash reaction product then with water.The washing step that water or similar water solvent (for example Shi Du salts solution) carry out has promoted that with poorly water-soluble or water-insoluble reaction product be other subsequent step that quaternary amine alkaloid alkylating agent changes into water-soluble cpds (for example salt).If the alkylating agent virulent material that is pair cell, then this alkylating agent preferably also is water miscible, just or at least one contact water resolve into water soluble component so that unreacted alkylating agent or hydrocarbonylation composition are removed from reaction mixture by washing.
Water-washing step can fully be simplified making processes, because this need not the explosion hazard of pure organic solvent (for example methyl ether) is taked complicated safe precaution measure, thereby can constantly simplify this process.And, also the nonideal water soluble component that exists in the reaction mixture is separated from reaction product and removed.It is shocking, people also find, product is changed into the step that the efficiency ratio of water miscible subsequent step washs with pure organic solvent and exceed 10 to 15 times, in this way, water-washing step has positive influence to the structure and the composition of reaction product, thereby has greatly improved the generation of ideal final product.
For example, this process can be used for alkaloid and requires the system cancer phosphorus of the compound mentioned in 1 to carry out alkylation reaction with containing AT 377 988, these phosphorus compounds as shown in Figure 3, Fig. 3 be especially suitable current application, particularly those have the application of acridyl.
The term of Shi Yonging " pilewort alkaloid " should refer to arbitrary member of selecting equally from comprise pilewort alkaloid, Oxychelidonine and the alkaloidal grouping of methoxyl group pilewort herein, unless require separately or from describe with implicit mode inference separately.The corresponding organic solvent that conforms to the present invention is that any alkaloid that is used to react dissolves in medicament wherein.For example, alkaloid can decompose in promoting and cause the organic solvent that alkaloid reacts, for example the solvent of selecting from the grouping by methyl chloride, methylene dichloride, chloroform, monochloroethane, ethylene dichloride and trichloroethane.
Alkaloidal alkylation reaction reacts under comparatively high temps, best results when solvent reaches boiling point.
After the washing, final reacting product is converted to water miscible.This can be according to the process described in AT 377 988 and the AT 354 644 by changing into water-soluble salt, especially changing into hydrochloride realizes, for example, in the sedimentary process of hydrochloride occurring or afterwards final reacting product is put into strong acid liquid or gas (for example HCl gas), or HCl solution is added in the organic solvent of reaction product of washing.As if by this acid treatment, separate the intermediate reaction compound that most of alkylating agents form between alkaloid and alkylating agent, stayed the alcaloid-derivatives of variation, wherein Kai Tou ternary nitrogen-atoms has been converted to quaternary nitrogen, in quaternary nitrogen, hydrogen atom group or the atomic group that results from alkylating agent are received restriction as the 4th ligand, and this is former
Son group preferably chooses from the grouping of being made up of methyl, ethyl and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide atomic group.For better understanding, it is the typical quaternary alkaloid reaction product of example with the pilewort alkaloid that formula subsequently (1) has been illustrated the present invention:
R1=methyl, ethyl, three (1-acridyl) phosphuret-(t)ed hydrogen sulfide, methyl-R2, ethyl-R2, belong to the R2 of three (1-acridyl) phosphuret-(t)ed hydrogen sulfide part
(see example 3) according to the present invention, carry out ultimate analysis from sedimentary one of them reaction product, as if---not bound by theory---stop the back at alkylation reaction (for example quaternary reaction) at least may be in sedimentary crystal by a part of alkylating agent of reaction mixture being carried out acid treatment and obtaining or decomposition compound by occlusion to a certain degree, or be adsorbed in firmly on the crystal by certain mode, therefore utilize the sedimentary purge processes of organic solvent washing such as ether and methylene dichloride it can not be removed.Yet provable, even mixed this material of following, whole effects of reaction product still can be brought into play fully.
The water-soluble salt of reaction product is fit to the application of injection solution.
In particular embodiment of the present invention, reaction and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide (CAS No.52-24-4) carries out, and in pharmacopeia, this material is also referred to as thiotepa.Other synonyms are ledertepa, Onco thiotepa, TESPA, tespamine, thiophosphamide, thio-TEPA, thiotriethylenephosphoramide, tifosyl, triaziridinylphosphine sulphide, N, N ', N " tri-1,2-ethanediylphosphorothioine triamide; N, N ', N " tri-1,2-ethanediylthiophosphoramide, tri-(ethyleneimino) thiophosphoramide; N, N ', N " triethylenethiophosphoramide, triethylenethiophosphorotriamide, m-triethylenethiophosphoramide, m-tris (aziridin-1-yl) phosphine sulphide, triethylenethiophosphoramide, tris (1-aziridinyl) phosphine sulphor, tris (ethylene-imino) thiophosphate, TSPA and WR 45312.
In particular embodiment of the present invention, alkaloid (arbitrarily, can be the total alkaloids of Chelidonium majus L.) extract in organic solvent with tris (1-aziridinyl)-phosphine sulphide (thiotepa) and final reacting product (arbitrarily, can be used as the mixture of compound) react, water is washed one time at least then.Because thiotepa is met water decomposition, reaction can be remained unconverted thiotepa residue later by this method and from organic phase, separate out.Method is that the organic solution that comprises intermediate reaction product (i.e. the compound that forms) between alkylating agent and alkaloid is washed with water for several times preferably, and it is reached capacity.Particularly desirable method is that washing is removed from reactant fully until the high toxicity thiotepa of remnants repeatedly.
In addition, utilize water will in medical applications, can cause reversed reaction even can cause some water-soluble toxic alkaloid of liver cirrhosis from the synthetic mixture, to be removed, or its concentration is reduced.Show by Ames test, but this reaction product mutagenesis prepared in accordance with the present invention.
When the total alkaloids extract from Chelidonium majus L. began, final reacting product was alkaloid and the alkaloidal mixture that contains the higher thiotepa reaction product of molecular mass, and the mixture of alkaloid degraded product.Because the building-up process difference, alkaloidal solubleness may change to some extent.Reaction product comprises these two the mixture of three (1-acridyl) phosphuret-(t)ed hydrogen sulfide reaction product of about 60～70% unreacted chelidonine and about 30～40%.
The tertiary amine alkaloid has been represented from the major portion of the alkaloid extract initial components of Chelidonium majus L. acquisition.For example, in the synthetic mixture, can be used as initial components and obtain following tertiary amine alkaloid: pilewort alkaloid, Protopine, stylopin, Yin not product, the high pilewort alkaloid of α, chelamidine, chelamine, L-Tocosamine, Chelidimerine, dihydrosanguinarine, Oxysanguinarine, Oxychelidonine and methoxyl group pilewort alkaloid.
After alkylation reaction stops, by water-washing step unreacted quaternary alkaloid (for example berberine) is removed from reaction mixture fully, and the alkaloid reaction product of unreacted water-soluble alkaloid and hydrocarbonylation is still stayed in the organic solvent.Character according to organic solvent that is used for alkylation reaction and/or alkylating agent, final intermediate reaction product may comprise the compound by thiotepa link, and one of them thiotepa molecule and one, two or three pilewort alkaloids, oxy-chelidonine or methoxy-chelidonine molecule are linked.In addition, it also may comprise the alcaloid-derivatives of hydrocarbonylation, wherein, alkaloid molecules such as pilewort alkaloid, oxy-chelidonine or methoxy-chelidonine link to each other with the linear alkyl atomic group of short chain on its quaternary nitrogen, especially link to each other with methyl or ethyl.And the compound of reaction of other hydrocarbonylations may be arranged in reaction mixture after alkylation reaction stops.
The reaction product that obtains from the total alkaloids of Chelidonium majus L. with thiotepa according to the present invention has the active spectrum of better medical treatment than the reaction product that obtains from the similar procedure of utilizing organic solvents such as diethylether to wash.At least some compound that exists in the reaction product of the present invention, especially quaternary amine pilewort alcaloid-derivatives can gather in cancerous tissue selectively,---cell growth inhibitor of all understanding than most of people---also can not attack healthy cell and by making cancer cell death come destruction of cancer cells, simultaneously.This utilizes said preparation to realize good medical tolerance, and can generally adapt to arrange because of heredity and cause dangerous higher medical treatment use of cancer cells diffusion even personal illness prevention to be used.It handles simple, and great reversed reaction can not occur in treatment doses.
The reaction product that obtains from the total alkaloids of Chelidonium majus L. with thiotepa has been showed the biological activity in the metabolic rule, it is not only applicable to prevention and treatment metabolic trouble, osteoporosis for example, but also be applicable to prevention and treatment rheumatosis, allergy, virus infection, epilepsy, multiple sclerosis, scar, dermatoma, wound after surgery and radiation injury.
The extract of Chelidonium majus L drying root can be used as the initial reactant of synthetics.These roots contain more alkaloid than leaf and stem.
Surprisingly, recent findings, when beginning from pilewort alkaloid, Oxychelidonine or methoxyl group pilewort alkaloid and as independent biological alkali source being, compare favourably according to the final reacting product of method of the present invention (for example referring to above example 3) acquisition quality of medical care of showing and the reaction product that activity can obtain with the total pilewort alkaloid alkylation reaction according to example 1 at least with it with commercial value.
Conventional medicine vehicle, especially liquor (for example injection liquid or immersion liquid), ointment, compress or sling are applicable to the medicine that comprises according to the reaction product of the present invention's preparation.
Figure of description
Fig. 1 is the HPLC collection of illustrative plates that all biological alkali composition of Chelidonium majus L. root place acquisition constitutes.
The preparations by HPLC finger printing of Fig. 2 for making according to example 1.
Fig. 3 is some phosphorus derivant that can be used as reactant.
Fig. 4 is the nuclear magnetic resonance spectrum of reaction product U-KRS.
Fig. 5 is the UV spectrum of reaction product U-KRS.
Fig. 6 is the UV spectrum of pilewort alkaloid salt hydrochlorate.
Fig. 7 is the mass spectral first part of reaction product U-KRS.
Fig. 8 is the mass spectral second section of the higher reaction product U-KRS of sharpness.
Fig. 9 is the mass spectrum of pilewort alkaloid salt hydrochlorate.
For further setting forth the present invention, enumerated following example.
Example 1:
A) alkaloidal extraction:
A. 25 gram alkaloid salt mixtures are placed water, pour in the separating funnel then.After adding 100 milliliters of methylene dichloride, shake separating funnel.Organic phase just can be separated, and then it is filled in the vial.
B. add 1N sodium hydroxide (pH8-9) to aqueous phase, muddy until occurring.After adding 100 milliliters of methylene dichloride, shake mixture.Organic phase just can be separated, and its methylene dichloride with step a is mixed mutually.Repeat this process, for example repeat 3 times.Filter and the mixing organic phase.
C. by adding sodium hydroxide the pH value of water is transferred to 10.After adding methylene dichloride, shake mixture.Organic phase just can be separated, and mixes with its filtration and with other organic phases.Utilize sodium hydroxide that the pH value of water is transferred to 13 now, utilize methylene dichloride fully to extract then.
D. the organic phase with these merging is evaporated to a kind of oily brown material of generation.
B) react with three (1-acridyl) phosphuret-(t)ed hydrogen sulfide:
The alkaloid resistates is dissolved in methylene dichloride, add three (1-acridyl) phosphuret-(t)ed hydrogen sulfide then.80 ℃ temperature this mixture is carried out refluxing in 2 hours.After being cooled to room temperature, clarify this reaction mixture.Filter then, in separating funnel, utilize 250 milliliters water washing to leach thing number time afterwards, for example 3 times or multipass more.
C) react with HCl
The solution of washing is packed in the glass beaker, add HCl gas and constantly stir it is reached capacity, hydrochloride just can separate out.Precipitate is filtered out,, after the drying it is dissolved in the water with the ether washing.
In mouse body, for the reaction product that example 1 makes, the LD50 value that is measured to is 485mg/kg.The machine of testing in mouse and rat shows, the hormone level that the product that makes according to invention has been regulated thymus gland, and in the animal body of those thymectomies, induce to have synthesized and have the material that similar thymosin alpha 1 active is arranged.This effect is based on dosage and obtains.Said preparation has increased peripheral blood T-lymphocytic quantity 50% (before 4.04 ± 0.43 * 109/l handles nearly, 6.24 ± 0.73 * 109/l handles the back), regulated the natural killer cell activity (198.20 ± 17.69% that penetrates antigenic humoral immunization reflection and spleen cell, control group is 71.50 ± 9.10%), and improved leukocytic Interferon, rabbit releasability in the animal experiment.Zooperal result is through clinical observation and definite.Therefore, also observing index of immunity on one's body at cancer patient is improved.
Use for prevention and immunology, the usage quantity of the preparation in the example 1 is 5 milligrams/70 kg body weight.For cancer therapy, it is relatively more suitable that per 20 kg body weight are used 5 milligrams of said preparations.
Example 2: the HPLC fingerprint
This mensuration is by ion pair reverse phase gradient chromatography and use a DAD detector to finish in 285 nanometers measurement absorbancy.Simultaneously, color atlas is drawn with reference to alkaloid by using.In addition, carry out a kind of HPLC-MSD and analyze, which peak value can draw by this analysis does not have outside these alkaloid scopes.Can obtain the HPLC collection of illustrative plates of Fig. 1 and 2 based on following experimental data:
Post: LiChrospher 60RP Select B, 5 μ m, 125 * 24mm ID
Elutriant: A) 200ml (acetonitrile)+800ml (water)+1.5g (hexane sulfonic acid)+0.3ml (85% phosphoric acid)
B) 900ml (acetonitrile)+100ml (water)+1.5g (hexane sulfonic acid)+0.3ml (85% phosphoric acid)
Gradient: 5 minutes isocratic elution 100% A;
24 minutes to 40% B
1 minute to 100% B
5 minutes 100% B;
5 minutes 100% A balances
Survey: UV lamp 285 nanometers
Wash-out flow rate: 1ml/min stopped after 35 minutes
Sampling volume: 10 μ l
Specimen preparation:
Extraction (Fig. 1) before the reaction: 25 milligrams of alkaloids are dissolved in 40 ml methanol by ultrasonic wave,, filter by membrane filter then near 50 milliliters.
Reaction product (Fig. 2): reaction product is converted into hydrochloride, is dissolved in the water and regulates pH between 2.5 and 6.5 with the concentration of 1mg/ml.
Example 3
According to the condition described in the example 1, pilewort alkaloid (Sigma) and three (1-acridyl) the phosphuret-(t)ed hydrogen sulfide (=thiotepa) with purifying of commercial applicability reacts.After alkylation reaction stops, the step of washing and utilizing HCl gas to transform, further handle final product according to following manner:
340 grams are soluble in water through the water-soluble reaction thing that HCl handle, and make its concentration near saturated, be placed on then in 8 ℃ the refrigerator.Occur spontaneous precipitation after a few hours, take out 264 milligrams of throw outs (hereinafter referred to as U-KRS).This throw out is made up of flaxen water-absorbent crystal, this crystalline melting range is very little, between 205-207 ℃ (referring to complete crystalline product), can send fluorescence azury during with the UV rayed of 366 nanometers, simultaneously can also see the xanchromatic track, orange and red fluorescence band.When utilizing this product of thin-layer chromatographic analysis, it does not move, and still is positioned at zero position (R
f=0), just track moves to R
f＞0.1.From nucleus magnetic resonance (NMR) spectrum (Fig. 4), can be clear that U-KRS comprise with pilewort alkaloid molecule in the similar aromatic ring of aromatic ring that contains.UV spectrum (Fig. 5) shows that absorbing maximum value is 148,155,160,205,230 and 282 nanometers, be very similar to the alkaloidal UV spectrum of pilewort (Fig. 6), being different from the pilewort alkaloid is that 240 nanometers and U-KRS appear at the spectrum in the peak value when being 230 nanometers separately.Nitrogen among this surperficial U-KRS is quaternary, and in the pilewort alkaloid is ternary.
From the mass spectrum shown in Fig. 7 and Fig. 8 (U-KRS) and Fig. 9 (pilewort alkaloid), can draw further detailed analysis, from the results of elemental analyses of U-KRS, disclose following things composition (table 1):
The elemental composition of table 1:U-KRS accounts for overall per-cent
| Element | Overall percentage (%) |
| C | 45.70 45.05 |
| H | 5,84 |
| N | 6.56 6.37 |
| O | 22.25 19.6 |
| P | 3.27 3.27 |
| S | 2.78 3.06 |
| Cl | 17.29 |
Following example has illustrated from the various application of the compound U-KRS of example 3 generations:
Example 4: by antitumour drug U-KRS the selectivity that cell in vitro increases is restrained
Material and method
Cell cultures is to contain 8% CO
2Damp atmosphere in temperature is 37-37.5 ℃ Roux bottle, carry out.The interflow is cultivated by the solution of the EDTA of 0.01% Regular Insulin and 0.2% and phosphate buffered saline (PBS) in addition isolating, and segregation ratio is 1: 5 to 1: 25.
Human endotheliocyte is handled with umbilical vein isolated by collagenase.The nutrient solution of endotheliocyte be the Vitrum AB of the foetal calf serum, 200 μ g/ml endotheliocyte growth factors and the 90 μ g/ml that utilize 15% thermal inactivation replenish M199.
Fluorescence microscopy
Cell is grown in 35 millimeters vessel, just can hatch in 30-60 minute when U-KRS is 100 μ g/ml.With the air emptying of nutrient solution, cell utilizes twice of PBS washing.Cover glass is installed on the cell, utilizes the confocal laser scanning microscopy of being furnished with argon laser to make fluorescence sensitization.] detect the light of being launched on the photomultiplier cell passage.Its signal just images on the video monitor that uses MRC 600 image processing softwares.
The result
1. measure in 20-40 μ g/ml U-KRS scope, supression is received in the growth of endotheliocyte about 55%.This concentration has been killed human osteosarcoma cell system.This two kinds of cell types assorted and having in normal cell susceptibility much at one.
2. owing to autofluorescence, therefore can in cell, detect U-KRS.Laser scanning microscope has shown that the height to U-KRS absorbs in malignant cell.
Example 5: U-KRS is as carcinostatic agent-oxygen-consumption
Material and method
In mouse body, test.Two to five control animals have all been injected the Ehrlich mouse ascites fluid suspension i.p. of 50 μ l, and this suspension is 8d old, are to absorb on one's body from the donations animal that grows up fully.This control group is not further treated.After cultivating, tumour injected 10 milligrams of U-KRS/ kilogram animal qualities at the belly of test group immediately.
Knot is adopted
After U-KRS intraperitoneal injection or subcutaneous injection, implant the mouse survival time of ascites tumour and be longer than the implantation animal for the treatment of without additive method.
By the mode at external use electrode the oxygen-consumption of ascites tumour suspension is tested discovery, oxygen-consumption increases slightly behind the interpolation U-KRS, but descends fast subsequently, is different from the contrast suspension that is not mixed with U-KRS.
Example 6: in rodent, use the improvement of the morphine analgesic effect of U-KRS.
Material and method
Animal: test on one's body male Albino Swiss mouse and male Wistar rats.
Treatment: with lowest dose level is that the U-KRS of 20mg/kg and 25mg/kg is expelled to respectively on Albino Swiss mouse and the Wistar mouse.
Process of the test: in each test, this four treated animal has all been injected) placebo, 2) morphine, 3) U-KRS, 4) U-KRS and morphine
The result:
The result shows, injects the drug effect that U-KRS and morphine have improved the narcotic analgesic medicine simultaneously.In the test that mouse carries out on one's body repeatedly, these two kinds of materials have produced analgesic effect, and sign shows that the pain relieving time length is longer.
This result shows, the U-KRS that uses in conjunction with morphine has been transformed into the nociception that institute described in testing with the susceptibility of experimental animal and reacts simultaneously.This result shows that U-KRS can interact with the analgesic drug product that is used for cancer.
Example 7: the death of the bimodulus sequencing cell that in malignant cell, produces by deutero-U-KRS.
Material and method
Use K562 erythroleukaemia clone, U-KRS produces complete crystallized form, and is dissolved in the water that concentration is 1.2mg/ml.
Utilize the DNA content of the K562 cell that propylidene and the analysis of fluidic cell meter show in the U-KRS of various concentration.
The result
The result of this research shows that U-KRS has produced bimodulus necrocytosis program, and wherein first form---natural death of cerebral cells---is by the Ca to the Quinidine sensitivity
2+-dependent K
+Passage causes; Second form---blister cell death---is by preventing that thereby microtubule from forming the generation polyploidy and causing.
Example 8: U-KRS is to synthetic etc. the influence in malignant cell of DNA, RNA and protein
Material and method
Be dissolved in indicating of 0.5 μ Ci in the 20 μ l media with indicating
3The thymidine of H; Urinate nucleosides, be dissolved in the L-LEU of 0.5 μ Ci in the 20 μ l media; The 20 μ l that are dissolved in 1.0 μ Ci in the 20 μ l media were placed in four different grooves of U-KRS concentration 2-4 hour.Before this, the human tonsilla cell of this clone, cavy liver cell, C1L liver cell, Muridae lymphoma, Muridae myelomatosis, Yoshida cell, two HeLa families, EsB-, EB, lymphoma, ZAC/1, P815 were grown up 24 hours in 37 ℃, the groove of 96 microtiter.
The WiDr cell began hatching in a different manner in 6 hours to 24 hours when U-KRS concentration is 1,4,8 and 14 μ g/ml.
The result
Fluorescence assessment has shown U-KRS and stronger magnetism at the cancer cells nucleus element in other cancer cells zone.Powerful and magnetism fast that fluorescence phenomenon may clearly illustrate that U-KRS appeared in tumour and transport zone.In the normal cell for the treatment of in the mode that cancer cells is had 100% supression effect of test so far, find no toxic action.
Example 9: U-KRS is to the influence of human heterograft
Material and method
From the human tumour heterogeneity graft, absorb tumour cell, and continuously it is transplanted in the nude mouse.These cells are used for external colony and form chemical examination.Utilize the medicine U-KRS of multiple concentration, the time at least one week of tumour cell constantly hatches.This utilizes six dissimilar realizations, and at each tumour colony is formed and to keep the score.Its drug effect is reported in this T/C (test/contrast).
The result
Many inhomogeneous tumours all produce responsive to the corresponding U-KRS of kind with the U-KRS test.As if this effects of tumors depends on the regenerative power of immune organ herein, stimulation and adjusting work may be finished by U-KRS.
Example 10: U-KRS is to the influence of human malignant's clone
Material and method
Use four different malignant clones: 1) mouse sarcomata; 2) female breast cancer; 3) human colon's cancer; 4) human melanoma
In nutrient solution, add U-KRS and PP9AA02 derivative.
After the irradiation, each lantern slide all makes 200 cells be coated with gold, in these cell one weeks of hatching, carries out painted to it then and counting.
The result
Result displayed shows that U-KRS and PP9AA02 derivative can act synergistically on human malignant's clone as toxicant herein.
Example 11: the G2/M that U-KRS brings out in the human epidermal cancerous cell line attracts and accent is died
Material and method
Main human keratinocyte and newborn dermatological specimens is isolated.Make epidermis thin slice trypsinize, and obtain single-cell suspension liquid by centrifuging.
The result
U-KRS restrains cell cycle progression according to dosage.
The U-KRS treatment influences the cell cycle distribution, and the trigger cell accent is died in A431 and ME180.
Cyclin, CDKs and CDKp27 restrain and can change after being expressed in of factor utilizes U-KRS to treat.
Example 12: the antimetabolic of U-KRS influence and to the oxygen metabolism of the mouse that has melanoma B-16 and the influence of energy metabolism
Material and method
Test on one's body at 133 C57B/6 male mouses.B-16 is transplanted on the correct integumentary musculature of every mouse.After this tumour transplatation the 10th day is divided into two groups with experimental animal.For first group, be that 0.05 milliliter, dosage are that the U-KRS of 1mg/kg is expelled in the venous sinus of eyes with volume: a shot is five times in two days.In the same way the disinfectant physiological solution is expelled in the venous sinus for second group.
The result
The result
Originally studies show that and U-KRS has been carried out first intravenous injection after one day, the oxygen therapy index in the muscle tissue has obtained remarkable improvement.PO
2The rate of change of level is increased to maximum value in the oxygen suction process, and after suction stopped, its speed was reduced to initial level from maximum value again.In the experimentation on animals group, taking preparation after one day, certain index of the oxidative phosphorylation in the liver mitochondrion is improved.As everyone knows, in the deterioration process, oxygen sucks and metabolism is restrained.In mouse, after 5 U-KRS injections, this kind damping effect weakens.In the control animal group, the oxygen density level in the muscle tissue and higher to the oxygen therapy speed of this tissue.Summarize the data obtained, perhaps can following fixed so conclusion, U-KRS can improve having the oxygen therapy of the tissue in the melanomatous mouse of B/16, also can consider to have restrained destructive effect the deterioration process from body bioenergetics angle.
Below many instance interpretation immunomodulatory and the metabolism adjusting function of U-KRS, pointed out that the treatment that U-KRS especially is suitable for irritated reflection, virus disease (HIV, the first and second the third livers, intestinal bacteria, influenza), osteoporosis, polyarthritis, psoriasis and other diseases or physical appearance handles.
Example 13: improve the scavenger cell anti-tumor activity by U-KRS
Material and method
BALB/c mouse obtains by brother-sister mating in the laboratory.By subcutaneous injection D1DMBA/3 tumour routine is transplanted among the BALB/c.After through cultivation 5 days, it is obvious that tumour becomes.
When Subcutaneous tumor is cultivated back 5 days, carry out handling in the body with U-KRS.Used three kinds to use routes in this process, i.e. intravenously, intraperitoneal and subcutaneous injection, these three experimental group, every group of at least 10 mouse have been injected the 0.15ml PBS solution of 5.0 μ U-KRS concentration.This dosage selects to be used for preliminary experiment.
The result
The speed of tumor propagation has obviously obtained reduction in the treated mouse.The mouse of injecting U-KRS does not show any deleterious medicine related side effects.
Example 14: U-KRS is to the external influence of normal people's lymphocyte phenotype
Material and method
The research is by finishing separating the lymphocyte research that obtains in 10 volunteer's peripheral bloods.These cells separate by Ficoll-Paque density gradient centrifugation and obtain.The cells whose development ability is measured by 0.1% trypan blue is painted, and the result is 95%.
Lymphocyte subpopulation formula is measured by immunofluorescence and is measured with respect to the monoclonal antibody quantity of total T cell, T-helper cell and T-suppressor cell.Then, cell uses FITC/conjugatedrabbit F/ab/2 fragments anti-mouse IgG to handle, and washs with PBS, and uses polyvinyl alcohol and glycerine to be fixed on the slice, thin piece.When making contrast, use PBS or normal mouse serum to replace monoclonal antibody.
The result
Present this studies show that U-KRS to the direct possibility of influence of T cell Ziren mouth quantity, and proved early stage observations, be exactly U-KRS be a kind of good immune exitation factor of cancer patient cellular immunization performance.
Example 15: U-KRS is to the mitogenesis attribute of human peripheral blood mononuclear cells
Material and method
Peripheral blood lymphocytes.Peripheral blood dilutes by the equal-volume PBS that comprises 1mM EDTA and pH7.5, and carries out layering on Histopaque 1077 tops.Centrifugal 30 minutes then with the rotating speed of 2000rmp.Collect upper layer and, wherein contain lymphocyte with RPMI tissue culturing medium washing 3 times.
The result
Discover, even adopt U-KRS to carry out the co of the pre-treatment meeting of certain very short time, compare, can obtain more cytositimulation with those situations of having only PHA to exist to a brute force of PHA mitotic division generation.And, find that also the short-term processing of PHA pair cell is essential for U-KRS generation co.
This research is showed, to handling the patient of worsening the stage in latter stage, can increase the quantity of circulating lymphocyte with U-KRS greatly.
Example 16: the immunological effect factor is by the adjusting of U-KRS to cells in vivo lytic activity and tumor propagation supression
Material and method
Tumour cell: mastocytoma P815 and AKR leukemia AKIL clone grow in the DMEM substratum, have 9.0% foetal calf serum, comprise penicillin and Streptomycin sulphate.
The result
Research is a kind of effective biological response modifier in the current body, makes the cell lysis activity of alloimmunization mouse spleen lymphocyte increase by 48 times.Disappear and measure in the serum by U-KRS being joined cell-mediated property, the cell lysis activity that spleen cell that IL-2 handles and peritonaeum slurry lymphocyte obtain has also obtained remarkable increase.
The result that these results and U-KRS also can be improved the cell lysis activity of spleen lymphocyte connects, and drawing in the body observed U-KRS result of treatment can mediate by immune stimulatory effector cell's cell lysis activity.
Example 17: U-KRS is to the influence of vivo and vitro immunity blood proterties
Material and method
96 Wistar rats are used for this research.The initial age of female mouse and male mouse was for 16 weeks.
U-KRS and PHA are detected by the lymphocytic #Hthymidine of T is detected, and assess the excitation index of from 0.01 to 20 μ g/ml different concns dosage with this.
The result
U-KRS has stimulated and has belonged to hematopoiesis and immune different subsystem.In this experiment, caused reticulocytosis, it may be that some stem cell stimulates or sign of the comprehensive activated of red blood corpuscle generation system.Just absolute white blood cell count does not change this point, can suppose the effect by U-KRS, only can strengthen the adjusting attribute, such as the confusion that can occur different sub-systems in this experiment.
Can also find that the stimulation that obtains in stimulated in vitro and these experiments has comparability, it mainly comprises the apoptosis of cancer cell.
Example 18: U-KRS is to the supression effect of ovalbumin antigenicity in the mouse body and the response of antiovalbumin IgE antibody
Material and method
(in the mouse of BALB/c * C57B1/6J), detected the supression ability of U-KRS at BALB/c and F1 to ovalbumin inductive sensitization.U-KRS is joined in the mouse, this mouse is in and contains in antigen (ovalbumin) and co-adjuvant (alum) mixture, the sensitization that can restrain mouse mainly is reflected in anti--OA IgE antibody response antigen induction histamine release lower and that separate on the mastocyte that obtains from activation mouse peritoneum chamber and reduces.U-KRS can react by rat allos PCA the antigenic influence of ovalbumin and detect in the supersensitivity reflection.
The result
The result shows, uses U-KRS and the response capacity of the OA that makes in mixture and anti--OA IgE antibody descends, and these anti--OA IgE production of antibodies are to be used for resisting in the mouse and in allos PCA reaction being fixed on the lip-deep natural OA of rat hypertrophy cell.Result hint, the OA pre-treatment of U-KRS can influence its antigenicity attribute and with the response capacity of the anti--OA IgE antibody that is used to resist natural IgE molecule.
Example 19: use the influence of U-KRS to early stage osteoporosis treatment
Material and method
U-KRS carried out intraperitoneal administration in per two days lasting six months with the dosage of 30 mg/kg to the female rat that suffers from the early stage osteoporosis of ovariectomy inductive.The operation second day, begin to carry out the U-KRS administration.Adopting the last of U-KRS long-term treatment, measure the humerus intensity of every rat and some performances of rat leg section.
The result
The result shows, can stop the leg section that physical strength reduces and ovariectomy causes of humerus to change by six months U-KRS treatments.
Example 20: the influence of U-KRS preparation popularity common cold virus and intestinal bacteria and streptococcus aureus
Material and method
The influenza virus of APR8/HON1/34 bacterial strain is cultivated 10 day time on the old hen embryo;
Use clinical material and the staphylococcus aureus strains 209P of intestinal bacteria as current application.290614 serial U-KRS preparations.
The result
Originally studies have shown that the U-KRS preparation has anti-infective performance in infected huge body.This influence is finished by the stimulation of the some parts in the host immune system, and this stimulates to destroy by microorganism or through the secondary of the cell of these infected by microbes and realizes.
Example 21: for the biological activity of influenza virus
Material and method
The A C-type virus C, Port-Chalmers 1/73 culture, antigenic H3N2 mutation.Each embryo is respectively with 1,10 and 100 EID
50Concentration is injected.U-KRS is dissolved in the Hanks solution again.
The result
Studies have shown that U-KRS has the behavior of pining down to course of infection.
Example 22: U-KRS is to the behavior of radiation effect
Material and method
The male mouse of the CBA/J of 16/20g body weight.
Mouse is carried out the short-term whole body gamma-radiation of scope from 6.0Gy to 7.5Gy.Use the CEGO device and use the semi-invariant of 8.75Gy to finish long-term radiation.
U-KRS carries out intraperitoneal administration with the dosage of 0.1,1.4 and 12 mg/kg body weight.
The result
Mouse is studied the ability that changes radiation effect by the U-KRS drug dose that uses 0.1 to 12 mg/kg.Discover that U-KRS has increased 50-60% for the survival rate of mouse 5.00 to the 7Gy radiation quantity, and do not exert an influence for the radiation quantity of 7.5Gy.Change drug dose and can not influence the radiating result.
The main result of research has found can change radiation effect when U-KRS uses prevention and methods of treatment at the same time at present.
Example 23: the effect of opposing ionizing rays
Material and method
The Brest carcinoma, colorectum gland cancer, malignant brain tumor and pancreatic cancer cell system.The U-KRS preparation.
Tested the effect of U-KRS pair cell survival in 0.2 μ G ML makes progress concentration range.Exposure duration is 1,3 and 24 hour, and after this cell is with phosphate buffered saline buffer with newly join nutrient solution and wash.
The result
U-KRS treats between the differentiation phase that has caused in the clone cell survival and the reduction of dose-dependently.Human tumour cell line to whole detections shows that they have different susceptibilitys for U-KRS, compares with the human fiber's archeocyte through 24 hours, and clone's survival rate has descended up to 100 times.
Claims (25)
1. alkaloid reactor product, at least comprise a kind of alcaloid-derivatives that the reaction of one or more alkaloids and alkylating agent obtains that comes from, here in quaternary ammonium derivative, contain the first uncle's base nitrogen, as the 4th aglucon, be connected with a hydrogen residue or come from a residue of alkylating agent, this residue is preferably selected for use from following groups: methyl, ethyl and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide residue, or come from the part of three (1-acridyl) phosphuret-(t)ed hydrogen sulfide.
2. the 1 alkaloid reaction product that obtains as requested, wherein at least a alcaloid-derivatives exists with the form of water-soluble salt, and preferably the form with hydrochloride exists.
3. the 1 alkaloid reaction product that obtains as requested, wherein alkaloid is at least the alkaloid in a kind of Chelidoniummajus of being present in L. medicinal herbs, is preferably the mixture of several or all biological alkali of Chelidonium majus L..
4. the alkaloid reaction product of any acquisition in 1 to 3 as requested, wherein alkaloid is selected from following material: Stylophorine, Protopine, stylopin, Yin product, homochelidonine, sanguinarine(e), chelamidine, chelamine, L-Tocosamine and Oxychelidonine.
5. the alkaloid reaction product of any acquisition in 1 to 4 as requested, wherein from dish alkali in the wrong, Oxychelidonine or methoxychelidonine as a kind of independent alkaloid source and exist.
6. the alkaloid reaction product of any acquisition in 1 to 5 as requested, wherein product comprises a kind of compound at least, comes from following material: the degradation production of unreacted tertiary amine alkaloid, unreacted alkylating agent and alkylating agent.
7. the alkaloid reaction product of any acquisition in 1 to 6 as requested, wherein product adopts following a kind of analysis means to show its result at least: the NMR spectrum among Fig. 4, the UV spectrum among Fig. 5, mass spectrum among Fig. 7 and 8 and the fundamental analysis in the table 1.
8. the alkaloid reaction product of any acquisition in 1 to 7 as requested can generate according to the technology that limits in requiring 11-22.
9. Stylophorine derivative, wherein the Stylophorine of natural generation exists according to following molecular formula (I) and with a kind of quaternary amine form,
Wherein, can be hydrogen or methyl or ethyl residue as the 4th aglucon.
10. require the Stylophorine derivative in 9 to present water-soluble form, preferably a kind of salt of strong acid especially is more preferably a kind of hydrochloride.
11. producing a kind of a kind of technology of alkaloid reaction product is limited to and requires to comprise in 1:
A) provide a kind of reaction mixture, comprise alkaloid and a kind of alkylating agent that uncle's base nitrogen is arranged on a kind of organic solvent and at least a chemical structure, and under the situation that organic solvent exists, allow at least a alkaloid and alkylating agent carry out an alkylation reaction, make and form at least a reaction product, wherein alkanisation occurs in alkaloid uncle base nitrogen position, therefore the nitrogen transformation of uncle's base can be become season base nitrogen;
B) after reaction finishes and obtains corresponding reaction product, carry out the once washing step at least, remove the solvable composition that is present in the reaction product with this with liquid solvent or water; And
C) making gaseous state or liquid strong acid come the processing reaction product, be preferably gas chlorination hydrogen or a kind of hydrochloric acid soln, be used at least a residue reaction product is changed into a kind of water-soluble form, especially is a kind of water-soluble salt.
12. require 11 technology, wherein in step c) after handling with acid, precipitation appears in reaction product, precipitates from organic solvent then and separates, and is more preferably with an organic solvent selectable being further purified.
13. require 11 or 12 technology, wherein alkylation reaction carries out under comparatively high temps, and is especially optimum in the boiling temperature of solvent.
14. any technology in 11 to 13 as requested, wherein at least a alkaloid is present in the Chelidoniummajus L. medicinal herbs, is preferably several or all biological alkali of Chelidonium majus L., can be used as biological alkali source and uses.
15. with any relevant flow process that requires 11-14, wherein alkaloid be from by pilewort alkaloid, Protopine, stylopin, Yin not product, the branch detail formed high pilewort alkaloid, sanguinarine(e), chelamidine, chelamine, L-Tocosamine and the Oxychelidonine chooses.
16. with any relevant flow process that requires 11-15, wherein pilewort alkaloid, Oxychelidonine or methoxyl group pilewort alkaloid can be used as independent biological alkali source.
17. with any relevant flow process that requires 11-16, wherein alkylating agent is promoting agent on physiological function, preferably says, should be cytotoxic agent.
18. with any relevant flow process that requires 11-17, the water decomposition of wherein alkylating agent water soluble, or chance is water-soluble composition.
19. with any relevant flow process that requires 11-18, wherein organic solvent promotes or causes the alkaloid reaction, this solvent is preferably selected from the grouping by methyl chloride, methylene dichloride, chloroform, monochloroethane, ethylene dichloride and trichloroethane.
20. with any relevant flow process that requires 11-19, wherein alkylating agent is three (1-acridyl) phosphuret-(t)ed hydrogen sulfide (CAS52-24-4).
21. with any relevant flow process that requires 11-20, wherein, reaction product comprises a compound of selecting at least from the grouping of being made of pilewort alkaloid, Oxychelidonine and methoxyl group pilewort alkaloid, wherein said compound has the quaternary nitrogen-atoms, the 4th ligand as this atom, hydrogen atom group or the atomic group that is derived from alkylating agent are received restriction, and this atomic group preferably chooses from the grouping of being made up of methyl, ethyl and three (1-acridyl) phosphuret-(t)ed hydrogen sulfide atomic group.
22. with any relevant flow process that requires 21, wherein three (1-acridyl) phosphuret-(t)ed hydrogen sulfide is as alkylating agent, wherein said residue is the part of said alkylating agent, owing to use acid to handle, therefore can arbitrarily form degradation production.
23. the reaction product of definition is as medicine or medicament in will requiring 1 to 10 any one.
24. the reaction product of definition is used for making the disease that prevention or treatment select from the grouping of being made up of following disease or the pharmaceutical cpd of physical appearance in will requiring 1 to 10 any one: virus infection, cancer, immunologic function disorder, metabolism disorder and radiation injury.
25. 24 used as requested, in this required, disease was to select from the grouping of being made up of allergy, osteoporosis, dermatoma, influenza infection, rheumatism, scar, wound after surgery, epilepsy and multiple sclerosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410045600 CN1706845A (en) | 2004-06-11 | 2004-06-11 | Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410045600 CN1706845A (en) | 2004-06-11 | 2004-06-11 | Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1706845A true CN1706845A (en) | 2005-12-14 |
Family
ID=35580974
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410045600 Pending CN1706845A (en) | 2004-06-11 | 2004-06-11 | Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1706845A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113853379A (en) * | 2019-05-13 | 2021-12-28 | 深圳艾欣达伟医药科技有限公司 | Fluorine-containing compound and anticancer medical application |
| CN114605422A (en) * | 2021-11-11 | 2022-06-10 | 广西师范大学 | A pair of enantiomer alkaloid dimer compounds, and preparation method and application thereof |
-
2004
- 2004-06-11 CN CN 200410045600 patent/CN1706845A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113853379A (en) * | 2019-05-13 | 2021-12-28 | 深圳艾欣达伟医药科技有限公司 | Fluorine-containing compound and anticancer medical application |
| CN113853379B (en) * | 2019-05-13 | 2024-04-30 | 深圳艾欣达伟医药科技有限公司 | Fluorine-containing compound and anticancer medical application |
| CN114605422A (en) * | 2021-11-11 | 2022-06-10 | 广西师范大学 | A pair of enantiomer alkaloid dimer compounds, and preparation method and application thereof |
| CN114605422B (en) * | 2021-11-11 | 2023-12-05 | 付训忠 | A pair of enantiomer alkaloid dimer compounds, and its preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1155571C (en) | Gamma-glutamyl and beta-aspartyl containing immunomodulator compounds and methods therewith | |
| CN1222526C (en) | Chlorophyll and bacteriochlorophyll esters, and their preparation | |
| CN109593711A (en) | Expand the amplification method and purposes of constant killer cell part | |
| CN1699558A (en) | Large-scale culture technology for killer cell for the induction of cell factor for hemopoietic stem cell by using bioreactor | |
| CN1198639C (en) | Immune enhancing compositions | |
| CN1835679A (en) | Process for producing human-origin immunocompetent cell | |
| CN1706845A (en) | Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid | |
| CN1532198A (en) | Quaternary chelidonine derivatives | |
| CA2593202A1 (en) | Quaternary alkaloid derivatives of chelidonium majus l | |
| CN1620305A (en) | Method of inducing apoptosis and compositions therefor | |
| CN102083806A (en) | 5-(4-methanesulfonyl-phenyl)-thiazole derivatives for the treatment of acute and chronic inflammatory diseases | |
| CN113024400A (en) | Colchicine derivative and preparation method and application thereof | |
| FI75802C (en) | Process for Preparation of Immunostimulants Using Bis and Polydisulfides | |
| CN1251678C (en) | Medicinal composition and tuchahoe extract for raising immunity | |
| CN1146432C (en) | Cell growth regulator | |
| CN1277537C (en) | Pharmaceutical compositions for immunosuppression, anticancer and antiviral treatment | |
| CN1582160A (en) | Novel herbal chemical composition for treatment of cancer | |
| CN85100625A (en) | The manufacture method of antineoplastic composite | |
| CN1566061A (en) | Novel natural drug effective region of rabdosiaexcisa total diterpene | |
| CN1654640A (en) | Superoxide dismutase composition and preparation method thereof | |
| CN1074219A (en) | New 10-hydroxyl pheophytin a | |
| CN1853724A (en) | Compound injection with probenecid, potassium and beta-lactam antibiotic and its use | |
| CN1486692A (en) | Plant extractive for treating cancer | |
| CN1477965A (en) | Pharmaceutical product for treatment and diagnosis of tumors and method for preparation of liqid free fraction of blood plasma | |
| CN1931186A (en) | Application of arsenous oxide in preparing medicine for treating embryo cell diseases sensitive to arsenide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |