CN104744478A - Compound for preventing and treating radiation injuries, single crystal as well as preparation method and application thereof - Google Patents
Compound for preventing and treating radiation injuries, single crystal as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a compound for preventing and treating radiation injuries, a single crystal as well as a preparation method and application thereof. The chemical structure of the compound is shown in a formula I specified in the description, a pharmacological activity test and an overall animal test prove that the compound in the formula I has good activity, especially the result of the overall animal test shows that the compound in the formula I has great anti-radiation activity and low toxicity, and therefore can be used for reducing the hematopoietic function injuries caused by radioactive or chemotherapeutic medicines, increasing the levels of blood cells, or carrying out adjuvant therapy for tumours. The single crystal of the compound in the formula I is further prepared from the compound disclosed by the invention, and is excellent in stability.
Description
Technical field
The present invention relates to pharmaceutical compound technical field, specifically, relate to a kind of compound, monocrystalline and its preparation method and application of preventing and treating radiation injury.
Background technology
Along with the progress of science and technology, nuclear energy is widely applied in every aspects such as numerous production, scientific research, medical treatment, also closely bound up with the daily life of the general population.Just because of this, to be subject to the chance of radiation increasing for human body.The worker etc. that the resident lived as high surrounding areas, nuclear industry field produce.Along with the fast development of medical skill, people have more and more many chances to touch ionizing rays, such as utilize radionuclide to the observation of disease and diagnosis, radioimmunoassay advancing of disease, radioscopy and tomoscan, in some oncotherapy, X-ray and gamma-rays is used to kill oncocyte.In addition, some special industries also can contact ionization radiation, as nuclear industry system to nuclear raw material exploration, exploitation, smelt and precision work, the production of nuclear fuel and reactor, use and study; New variety, vegetable and fruit storage, grain stocking are cultivated in agricultural sector's radiation; The various accelerators of industrial sector, ray generator and electron microscope, electronics speed welding machine, colour kinescope, high-voltage electrical tube and space probation etc.Excessively for a long time be subject to radiation effect body will be caused to produce a series of damage, as dysfunction, the immune system dysfunction of oxidativestress damage, hemopoietic system, even DNA splitting of chain, transgenation, Chromosome recombination, cell transformation and necrocytosis etc.Therefore, research radiation injury means of prevention, finds effective protective agents, has caused the common concern of people.
The chemical protection of current radiation reaches mainly through the radio-protector and some crude substance applying synthesis the object preventing or alleviate radiation injury.Radio-protector refers to that body or a certain biosystem are subject to before and after ionizing rays, gives in early days to alleviate its radiation injury, the chemicals promoting it to recover or compound.In recent years, ammonia sulfenyl compounds, cytokine, hormones, natural drug etc. are all used to the chemical protection of radiation injury.Wherein, amifostine is that FDA ratifies widely used radio-protector, but amifostine exists the problems such as drug effect difference, side effect be many.Other radio-protector does not all reach clinical application degree.Clinical in developing low toxicity, curative effect preferably radio-protective new drug.
Summary of the invention
The object of the present invention is to provide a kind of compound and the crystal thereof of preventing and treating radiation injury.
The invention provides a kind of compound preventing and treating radiation injury, the chemical structure of described compound is such as formula shown in I:
The invention provides a kind of preparation method of formula I, comprise the following steps:
A) 3-(6 is got, 7-dihydro-1-methyl-7-oxo-3-propyl group-1H-pyrazoles [4,3-d] pyrimidine-5-yl)-4-oxyethyl group-benzene sulfonyl, be dissolved in chloroform, ice bath, stirring and dissolving, add triethylamine, 4-methylimidazole, stir, TCL monitors reaction, run plate and lose main raw material, stopped reaction;
B) reaction solution adds saturated sodium bicarbonate solution washing, then with sodium chloride solution washing, separates organic phase, dry;
C) filtrate evaporate to dryness, obtains oily matter, adds isopropyl ether, acetonitrile, and clarification, stirring at room temperature, slowly separates out white solid, obtains described formula I.
The invention provides a kind of monocrystalline of formula I, there is characteristic peak at the following 2 θ angles of described monocrystalline in powder x-ray diffraction collection of illustrative plates: 13.5 ± 0.1 °, 15.2 ± 0.1 °, 20.5 ± 0.1 °, 21.8 ± 0.1 °, 22.8 ± 0.1 °, 25.5 ± 0.1 °, 27.9 ± 0.1 °.
In another preference, there is characteristic peak at the following 2 θ angles of described monocrystalline in powder x-ray diffraction collection of illustrative plates: 13.5 ± 0.1 °, 15.2 ± 0.1 °, 20.5 ± 0.1 °, 21.8 ± 0.1 °, 22.8 ± 0.1 °, 25.5 ± 0.1 °, 27.9 ± 0.1 °, 31.0 ± 0.1 °, 33.1 ± 0.1 °.
The invention provides a kind of monocrystalline of formula I, described monocrystalline characterizes as follows:
Crystallographic system: triclinic(crystalline)system
Spacer: P-1
Unit cell dimension:
α＝88.330(4)°
β＝77.426(4)°
γ＝79.072(4)°
Volume:
Z＝2。
The invention provides the preparation method of the monocrystalline of described formula I, comprise the following steps: modus ponens I, add methylene dichloride and normal hexane, backflow, clarification, place, slowly crystallization, obtains monocrystalline.
In another preference, the preparation method of the monocrystalline of described formula I comprises the following steps: modus ponens I, adds methylene dichloride and normal hexane, backflow 1-10 hour, and clarification, places 1-30 hour, slowly crystallization, obtain monocrystalline; Described methylene dichloride and formula I ratio are 10-80ml:1g, and described normal hexane and formula I ratio are 5-70ml:1g.
The invention provides the pharmacy acceptable salt of the monocrystalline of described formula I or formula I, described salt is inorganic acid salt or organic acid salt, and described inorganic acid salt is hydrochloride, vitriol, phosphoric acid salt, diphosphate, hydrobromate or nitrate; Described organic acid salt is acetate, maleate, fumarate, tartrate, succinate, lactic acid salt, tosilate, salicylate or oxalate.
The invention provides a kind of pharmaceutical composition, described pharmaceutical composition contains treatment the formula I of significant quantity, the monocrystalline of formula I or described pharmacy acceptable salt, and the pharmaceutically acceptable carrier of surplus.
The invention provides formula I, the monocrystalline of formula I or the pharmaceutical applications of described pharmacy acceptable salt, for the preparation of the medicine of control radiation injury.
Present invention also offers formula I, the monocrystalline of formula I or the pharmaceutical applications of described pharmacy acceptable salt, for:
A) red corpuscle suppressing radiation to cause and leukocyte count object reduce,
B) red corpuscle suppressing radiation to cause and the reduction of platelet content,
C) alleviating of the spleen weight preventing radiation from causing,
D) medullary cell is improved to the tolerance of radiation, or
E) lost of life suppressing radiation to cause.
The invention has the advantages that:
1, formula I is prepared first, and tested by pharmacologically active and whole animal test, confirmation formula I has good cytoactive, particularly whole animal test-results display type I has fabulous radioprotective activity, and toxicity is low, therefore can be used for alleviating hemopoietic function damage that radiation or chemotherapeutics cause, elevating blood cell levels, or tumor aid treatment.
2, the monocrystalline of formula I is obtained first, good stability.
Accompanying drawing explanation
Fig. 1. be the syntheti c route figure of formula I.
Fig. 2 .SDLF-510 is on the impact of irradiated mice peripheral hemogram.60Co-gamma-rays (7.0Gy) whole body exposure, gives SDLF-510100mg/kg after SDLF-510 group irradiation, administration 3 days; N=20; Result * P<0.05, * * P<0.01vs Nor or Rad.Nor: Normal group, Rad: irradiation control group, SDLF-510:SDLF-510 compound group.
Fig. 3 .SDLF-510 is on the impact of irradiated mice body weight, spleen and thymus gland.60Co-gamma-rays (7.0Gy) whole body exposure, dosage 100mg/kg, administration after irradiation, administration 3 days.N＝20。*P<0.05，**P<0.01vs Nor or Rad。Nor: Normal group, Rad: irradiation, DLF-510:DLF-510 compound group.
Fig. 4 .SDLF-510 affects irradiated mice hemopoietic function of bone marrow.60Co-gamma-rays (7.0Gy) whole body exposure, dosage 100mg/kg, administration after irradiation, administration 3 days.7th day bone marrow smear record bone marrow nucleated cell (karyocyte), marrow granule (bone narrow particles) (Wright-Giemsa × 400), marrow HE dyes and calculates hematopoiesis area (hematopoietic area).Select to be coated with counting at least 5 high power lens visuals field, lamellar body tail cell plain cloth place to average.Count at least 5 high power lens visuals field to average, result represents with mean ± standard deviation (mean ± SD), and ANOVA checks.N＝20。*P<0.05，**P<0.01。Nor: Normal group, Rad: irradiation, SDLF-510 compound group.
Fig. 5 .SDLF-510 reduces irradiation apoptosis in bone marrow cells.Tunel (× 200) selects counting each at least 5 middle high power lens visuals field in smear cells even plain cloth place to average, and calculates apoptosis ratio, and result represents with mean ± standard deviation (mean ± SD), and ANOVA checks.N＝3。**P<0.01。Nor: Normal group, Rad: irradiation control group, SDLF-510:SDLF-510 compound group.
Fig. 6. the powder x-ray diffraction spectrogram of the crystal of formula I, the longitudinal axis represents peak intensity (cps), and transverse axis represents diffraction angle (2 θ [°]).
Fig. 7. concrete each peak position data.
Fig. 8. the monocrystalline molecular structure of formula I.
Embodiment
The present inventor has prepared a kind of compound such as formula the novel structure shown in I through research, and the formula I of confirmation has the effect of control radiation injury, prepares the crystal of formula I simultaneously, finds that this crystal has good stability.Complete the present invention on this basis.
The present invention provide firstly a kind of chemical structure such as formula the compound shown in I, or this compound pharmacy acceptable salt, crystal formation, prodrug, solvate or hydrate:
This formula I chemistry is by name: 5-(2-oxyethyl group-5-((4-methyl-1 H-imidazole-1-group) alkylsulfonyl) phenyl)-1-methyl-3-propyl group-1H pyrazoles [4,3-d] pyrimidine-7 (6H)-one (code name: SDLF-501);
English language Chemical is called:
5-(2-ethoxy-5-((4-methyl-1H-imidazol-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one。
As chemical structural formula conflicts mutually with name, be then as the criterion with chemical structural formula.
The present invention also comprises corresponding all pharmacy acceptable salts of above-claimed cpd, crystal formation, prodrug, solvate or hydrate.These salt can by part positively charged in compound with there is the electronegative of opposite-sign formed; Or formed with positive charge by part electronegative in compound.
The preparation of this compound can be realized by the route of accompanying drawing 1.The compound of synthesis can be further purified further by modes such as column chromatography, high performance liquid chromatography or crystallizations.
The purified compound more than obtained, by adding methylene dichloride and normal hexane, backflow, clarification, place, slowly crystallization, single crystal compound.
As used herein, " room temperature " refers to temperature range well known in the art, is preferably 20-30 DEG C.
crystal
As used herein, term " crystal " refers to that molecule or atom recombination thing are the solid of particular arrangement form.Term " monocrystalline " (single crystal), namely the particulate of xln inside is arrange regularly, periodically at three-dimensional space, the entirety of crystal is made up of the same space grid on three-dimensional in other words, and in whole crystal, particle is arranged as long-range order in space.When X-ray can produce a series of diffraction through crystal, use x-ray diffractometer can obtain powder x-ray diffraction collection of illustrative plates in the lab, in collection of illustrative plates, the intensity of different diffracted rays and each diffracted ray is determined by the atomic group of a fixed structure, the diffracting spectrum that the crystal of different structure produces is different, and X-ray diffracting spectrum therefore can be used to determine the structure of crystal.
The method that the X-ray diffracting spectrum measuring crystal adopts is known in the art.
The monocrystalline of formula I of the present invention has specific crystal habit, and its powder x-ray diffraction figure has specific diffractive features peak.
Particularly, the monocrystalline of formula I of the present invention has following a series of feature 2 θ angle diffraction peak in powder x-ray diffraction collection of illustrative plates: 13.5 ± 0.1 °, 15.2 ± 0.1 °, 20.5 ± 0.1 °, 21.8 ± 0.1 °, 22.8 ± 0.1 °, 25.5 ± 0.1 °, 27.9 ± 0.1 °; Preferably, feature 2 θ angle diffraction peak is: 13.5 ± 0.1 °, 15.2 ± 0.1 °, 20.5 ± 0.1 °, 21.8 ± 0.1 °, 22.8 ± 0.1 °, 25.5 ± 0.1 °, 27.9 ± 0.1 °, 31.0 ± 0.1 °, 33.1 ± 0.1 °; More preferably, it has the powder x-ray diffraction collection of illustrative plates substantially consistent with accompanying drawing 6.
The monocrystalline of formula I of the present invention possesses following parameter:
Crystallographic system: triclinic(crystalline)system
Spacer: P-1
Unit cell dimension:
α＝88.330(4)°
β＝77.426(4)°
γ＝79.072(4)°
Volume:
Z＝2。
The monocrystalline molecular structure of formula I of the present invention as shown in Figure 8.
The preparation method of the monocrystalline of formula I is preferably: add methylene dichloride and normal hexane, backflow, clarification, and place, slowly crystallization, obtains monocrystalline.But those skilled in the art have, and motivation attempts using toluene, normal heptane equal solvent carries out crystallization.
pharmaceutical composition
The present invention also relates to the pharmaceutical composition or pharmaceutical preparation that include xln formula I simultaneously, they can be made into such as to be suitable for various forms that is oral or injection, for control radiation injury, particularly alleviate radiation or chemotherapeutics cause hemopoietic function damage, elevating blood cell levels, or for tumor aid treatment, comprise particularly: red corpuscle a) suppressing radiation to cause and leukocyte count object reduce, b) red corpuscle suppressing radiation to cause and the reduction of platelet content, c) alleviating of the spleen weight preventing radiation from causing, d) medullary cell is improved to the tolerance of radiation, or e) suppress radiation to cause the lost of life, but be not limited only to this.Preferred medicinal preparations formulation can be tablet, capsule, microgranules and suspensoid.In pharmaceutical formulation involved in the present invention, except xln formula I, also comprise pharmaceutically acceptable carrier, excipient and/or thinner.Representational example comprises (but being not limited to):
One or more weighting agents, as Microcrystalline Cellulose, lactose, sucrose, starch, treated starch, seminose, dextran, calcium carbonate, phosphoric acid salt, vitriol;
One or more binding agents, as lactose, starch, treated starch, dextran, Microcrystalline Cellulose, sucrose, polyether glycol, hydroxypropylcellulose, Vltra tears, Natvosol, methylcellulose gum, carboxymethyl cellulose, gelatin, polyvinylpyrrolidone, magnesium aluminum silicate;
One or more disintegrating agents, as cross-linked polyvinylpyrrolidone, crosslinked carboxymethyl fecula, starch, Microcrystalline Cellulose etc.
In addition, if necessary, tensio-active agent and pigment can also be comprised in the formula of pharmaceutical composition.
Treatment significant quantity (that is: can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts) compound of the present invention (be used for the treatment of the carrier of administration with medically acceptable carrier, they itself are not necessary activeconstituents, and after using, there is no undue toxicity) and can pharmaceutical preparation be formed, these pharmaceutical preparations can be prepared into oral preparations, injection, tablet, powder preparation, capsule, dispersible tablet, sustained release preparation etc.
The consumption of the composition of the present invention for the treatment of significant quantity is between 0.001-500mg/kg body weight/day, and any consumption within above-mentioned scope is all significant quantity of the present invention.Described " treatment significant quantity " can be used for single drug or the drug combination treatment of relative disease.It will be appreciated by those skilled in the art that the consumption when actual administration can higher or lower than above-mentioned dosage range.Can by the impact of factors for " the treatment significant quantity " of a certain object (as Mammals-people) and concrete treatment plan, comprise the drug activity of compound used therefor or its prodrug, age of administration object, body weight, generalized case, sex, diet, administration time, disease susceptibility, disease process and accept the judgement etc. of doctor for medical treatment.
Formula I or its composition can by the administrations such as oral, intravenously, intramuscular, subcutaneous, nasal cavity, internal rectum.Solid carrier is as starch, lactose, phosphoric acid glycol, Microcrystalline Cellulose, sucrose and white bole, and liquid carrier is as sterilized water, polyoxyethylene glycol, nonionic surface active agent and edible oil (as Semen Maydis oil, peanut oil and sesame oil), as long as be applicable to the characteristic of activeconstituents and required specific administration mode.In pharmaceutical compositions, normally used adjuvant also can advantageously be included, and e.g., seasonings, pigment, sanitas and antioxidant are as vitamin-E, vitamins C, BHT and BHA.
These active compounds also can parenteral or intraperitoneal administration.Also solution or the suspension of these active compounds (as free alkali or pharmacy acceptable salt) can be prepared in the water being suitably mixed with tensio-active agent (as hydroxypropylcellulose).Also can prepare dispersion liquid in glycerine, polyoxyethylene glycol and the mixture in oil thereof.Under conventional storage and working conditions, contain sanitas in these preparations to prevent microbial growth.
The medicament forms being applicable to inject comprises: aseptic aqueous solution or dispersion liquid and aseptic powder (for extemporaneous preparation of sterile injection liquid or dispersion liquid).In all situations, these forms must be aseptic and must be that fluid is to be easy to syringe displacement fluids.Must be stable under conditions of manufacture and storage, and microorganism must be able to be prevented as the pollution of bacterium and fungi and impact.Carrier can be solvent or dispersion medium, wherein containing, for example water, alcohol, their suitable mixture and vegetables oil.
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
The preparation of embodiment 1 formula I (code name SDLF-501)
1, the preparation of intermediate 3-(6,7-dihydro-1-methyl-7-oxo-3-propyl group-1H-pyrazoles [4,3-d] pyrimidine-5-yl)-4-oxyethyl group-benzene sulfonyl
By reference
Bell AS,Brown D,Terrett NK.Pyrazolopyrimidinone antianginal agents[P].EP:463756A1.1991-06-07；
The method preparation of Bell AS, Terrett NK.Pyrazolopyrimidinones antianginal agents [P] .EP:526004,1992-07-02..
The nucleus magnetic hydrogen spectrum of product and mass-spectrometric data are:
1H NMR(600MHz,DMSO)δ7.89(d,J＝2.2Hz,1H),7.74(dd,J＝8.6,2.2Hz,1H),7.13(d,J＝8.7Hz,1H),4.18(s,3H),4.15(q,J＝6.9Hz,2H),2.81(t,J＝7.5Hz,2H),1.74(dq,J＝14.8,7.4Hz,2H),1.33(t,J＝6.9Hz,3H),0.95(t,J＝7.4Hz,3H).HRMS[M+H]411.09。
2, the preparation of SDLF-501
Get 3-(6,7-dihydro-1-methyl-7-oxo-3-propyl group-1H-pyrazoles [4,3-d] pyrimidine-5-yl)-4-oxyethyl group-benzene sulfonyl 10.5g (25mmol), be dissolved in chloroform 150ml, ice bath, stirring and dissolving, add triethylamine 5.05g (50mmol), 4-methylimidazole 2.5g (30mmol) is dissolved in chloroform 25ml, after clarification in impouring reaction flask, stirs, TCL monitors reaction, developping agent: methylene dichloride: ethyl acetate=8:2 (volume ratio), runs plate and loses main raw material, stopped reaction.
Reaction solution moves in 250ml separating funnel, adds saturated sodium bicarbonate solution 15ml and washs, and reaction solution washs secondary with sodium chloride solution 20ml again, separates organic phase, adds anhydrous magnesium sulfate 3g drying.Filter, remove anhydrous magnesium sulfate, filtrate evaporate to dryness, obtain oily matter 9g.Add isopropyl ether 100ml in oily matter, then add acetonitrile 3ml, clarification, stirring at room temperature, slowly separate out white solid 5.5g.Solid adds methylene dichloride 100ml, heating, and clarification, adds activated carbon 1g, reflux 10 minutes, filters, and filtrate is placed, slowly crystallization, and crystallization is filtered, and obtains 3.8g.Productive rate 33.3%.
The nucleus magnetic hydrogen spectrum of product and mass-spectrometric data are:
1H NMR(600MHz,DMSO)δ12.26(s,1H),8.27(s,1H),8.19(d,J＝1.3Hz,1H),8.19–8.16(m,1H),7.47(s,1H),7.41(d,J＝8.8Hz,1H),4.22(q,J＝7.0Hz,2H),4.18(s,3H),2.79(t,J＝7.5Hz,2H),2.08(s,3H),1.78–1.71(m,2H),1.32(t,J＝6.9Hz,3H),0.95(t,J＝7.4Hz,3H).HRMS[M+H]457.166。
The control radiation injury test of embodiment 2 SDLF-501
Radiation injury will cause body to produce a series of damage, as dysfunction, the immune system dysfunction of oxidativestress damage, hemopoietic system, and even DNA splitting of chain, transgenation, Chromosome recombination, cell transformation and necrocytosis etc.According to the feature of radiation injury; in this test; our high spot reviews formula I, on the impact of the visceral organ injuries such as irradiated mice survival rate, body weight, bonemarrow nucleated cells number amount, marrow hemopoiesis area, spleen, peripheral hemogram and other function indexs of correlation, have rated the provide protection of SDLF-501 to irradiated mice.
1 material
1.1 laboratory animal
ICR mouse, male, about body weight 21-26g, purchased from Shanghai Si Laike animal scientific & technical corporation.Raise in The 2nd Army Medical College IVC system.Experimental session, keeps Animal House room temperature at about 22 DEG C, relative humidity about 70%, early 8 extremely late 8 automatic illuminatings.Animal ad lib, freely drinks water.
1.2 main agents and medicine
1.2.1 integral experiment
Physiological saline Beijing Double-Crane Pharmaceutical Co., Ltd
Sheng Gong bio tech ltd, Xylo-Mucine Shanghai
This seminar of SDLF-501 synthesizes
1.2.2 Pathological experiment
1.2.3 cell experiment
1.3 major experimental equipment
2 experimental techniques
2.1 animal grouping and irradiation
After male ICR mouse conforms, be divided into three groups at random: 1) blank group, 2) irradiation control group, 3) SDLF-501 group, every treated animal experimentally requires 20.60Co-gamma-rays (7.0Gy) whole body exposure animal is used at Second Military Medical University, PLA's Radiation Center.
2.2 animals administer
Irradiation administration after 1 hour, adopt gastric infusion, solvent uses 0.5% Xylo-Mucine, and administration volume is 20ml/kg.Blank group is with 0.5% Xylo-Mucine gastric infusion; Irradiation control group is with physiological saline gastric infusion; Administration group, SDLF-501100mg/kg.Successive administration 3 days.
2.3 SDLF-501 are on the impact of irradiated mice survival rate
Administration 3 days after irradiation, Continuous Observation 20 days, record animal state as free activity, hair, body weight, and adds up death condition.
2.4 SDLF-501 are on the impact of irradiated mice body weight and peripheral hemogram
Experiment is divided into blank group, irradiation control group, SDLF-501 (100mg/kg) treatment group.Often organize each 20 mouse, give said medicine process and 7.0Gy gammairradiation, and getting blood according to latter 7 days posterior orbits of weighing, automatic blood cell calculating instrument detects peripheral hemogram.
2.5 SDLF-501 weigh and organ index impact irradiated mice chest gland weight, spleen
Grouping and process are with 2.4.After weighing according to latter 7th day, cervical dislocation is put to death, and gets thymus gland, spleen is weighed, and calculates thymus gland, spleen coefficient.
2.6 SDLF-501 are on the impact of irradiated mice medullary cell
Grouping and process are with 2.4.After weighing according to latter 7th day, cervical dislocation is put to death, and gets femur, cuts off the 2nd femur, with tweezers folder femur to extrude marrow, is placed on a slide glass, smear.One, every animal smear.(1) treat that dye smear lies against on staining rack by what dry; (2) with dropper, dye liquor is dripped on smear, cover whole smear, place 1 minute; (3) add the phosphoric acid buffer of equivalent, mix with dye liquor, dye under room temperature 20 ~ 30min; (4), at the end of dyeing, with distilled water, the dye liquor on smear is directly washed out; (5) dry or dry slide; (6) mounting; (7) microscopic examination.
The impact that 2.7 SDLF-501 change irradiated mice marrow protection
Grouping and process are with 2.4.Get the capable pathological staining of bilateral femur and observe bone marrow smear change.Marrow paraffin section HE dyeing process and step: (1) draws materials the same.Femur is inserted special pathology embedding frame, numbering is placed in paraformaldehyde liquid fixing, and the set time is no less than 24h.(2) decalcification: according to forefathers' experience, Plank Rycho decalcifying Fluid is desirable to bone tissue's decalcification effect.Its character is gentle, little to bone destruction, can holding structure complete, clear contrast of dyeing is obvious, and after decalcification need not in and program directly can enter dewatering process.By decalcification under the osseous tissue sample room temperature that fixes 2 ~ 4 days, wherein at least change 1 decalcifying Fluid, to float with osseous tissue and pin can pass through tissue shows that decalcification is complete.(3) dewater, transparent, waxdip, embedding: dehydration adopts ascending gradient alcohol, from 75%, 85%, 95% to dehydrated alcohol, alcohol at different levels does not last 1 ~ 2h not etc., the then transparent about 1h of dimethylbenzene.(4) section, paster, roasting sheet.(5) dewaxing, rehydration, dyeing.(6) microscopy after rear neutral gum mounting is dried.(7) utilize Image professional software and net form micrometer point count to measure percent of volume shared by hemopoietic tissue in marrow protection section, every femur sample at least get 5 high power fields take pictures measure after average.
2.8 cell levelss determine the provide protection of SDLF-501 to hemopoietic function of bone marrow obstacle
Experiment is divided into blank group, irradiation control group, SDLF-501 (100mg/kg) treatment group.Get normal ICR mouse primary medullary microeirculation, at the parallel 60Co-gamma-ray irradiation of cellular water, irradiation dose 8Gy.Treatment group gives SDLF-501 (final concentration is 1mM) and intervenes, and irradiation control group gives with measuring PBS, and after administration, the radiation-induced apoptosis of 24h capable 60Co-γ, utilizes apoptosis kit detection cell apoptosis after irradiation in 24h.
2.9 data statisticss and analysis
In experimental result, measurement data represents with mean ± standard deviation (mean ± SD).Comparison between two groups adopts independent t test method.Relatively select one-way analysis of variance (ANOVA) between many groups, adopt LSD to carry out multiplely comparing between two.The comparison of survival rate adopts Kaplan-Meier analytical procedure, and Log-Rank is for evaluating the difference of survival curve.P < 0.05 thinks that difference has significance,statistical.SAS statistical software is adopted to process.
3 results
3.1 SDLF-501 are on the impact of irradiated mice state and survival rate
Each 20 of two control group (blank group and irradiation control group) animals, SDLF-501 treated animal 20, ICR mouse is after 7.0Gy 60Co-γ irradiation, from 5 day beginning mouse, control group (blank group and irradiation control group) animal gradually occurs that lazyness is dynamic, hair is withered, appetite is poor, body weight declines gradually; From 8 day beginning mouse, SDLF-501 treated animal gradually occurs that lazyness is dynamic, hair is withered, appetite is poor, body weight declines gradually; Afterwards the body weight of SDLF-501 group mouse experienced by and first decline after rise phenomenon gradually.After administration 20 days, control group (blank group and irradiation control group) animal is all dead, administration group (SDLF-501 group) animal dead 8, compare with control group (blank group and irradiation control group), the survival time significant prolongation of SDLF-501 treated animal.Within 20 days, survival rate is 60%.
3.2 SDLF-501 is on the impact of irradiated mice peripheral hemogram
Compare with intact animal (i.e. blank group), model group (irradiation control group and SDLF-501 group) murine interleukin (WBC), red corpuscle (RBC), thrombocyte (PLT), reticulocyte ratio (IRF) etc. all have and reduce in various degree.But go up to some extent after SDLF-501 group.The red corpuscle that SDLF-501 causes for radiation and leukocyte count object reduce obvious restraining effect (Fig. 2).
3.3 SDLF-501 are on the impact of irradiated mice Spleen coefficient and thymus gland coefficient
Compare with intact animal (i.e. blank group), irradiation model group (irradiation control group and SDLF-501 group) the weight of animals has decline.Spleen, thymus gland coefficient reduce.This experiment finds, excessively alleviating of the spleen weight that SDLF-501 can prevent ray from causing.After radiation injury, the one of the main reasons of hematopoietic disorder is the damage of ray to the hemopoietic stem cell maintaining hemopoietic function of relying, and the damage of hemopoietic stem cell is the key factor determining body death.This experiment finds that SDLF-501 group is by according to endogenous mouse Spleen nodes comparatively control group showed increased, points out SDLF-501 to protect and is subject to according to mouse hematopoietic stem cell (Fig. 3).
3.4 SDLF-501 are on the impact of irradiated mice medullary cell
Got blood examination after irradiation to survey in 7 days, compared with normal group (i.e. blank group) mouse, after irradiation, mouse (irradiation control group and SDLF-501 group) routine blood indexes (except red corpuscle) all has decline, obviously, bonemarrow nucleated cells number and hematopoiesis area also obviously decline the damage of marrow granule.SDLF-501 group mouse bone marrow cells smear shows that frangments smear significantly increases, and marrow granule form is recovered to some extent, and pathology shows that hemopoietic tissue area has remarkable increase (* * P < 0.01, * P < 0.05).The above results shows that mouse is after SDLF-501 process, and its medullary cell has larger tolerance (Fig. 4) to radiation.
3.5 SDLF-501 alleviate myeloid element radiation damage (cell levels)
Cultivate primary bone marrow cells (source ICR mouse) 2 days, give PBS (irradiation control group), SDLF-501, final compound concentration 1mM respectively, 24h expert 60Co-γ irradiation after administration, cell death inducing, irradiation dose 8Gy.After irradiation 24h, TUNEL detects apoptosis.Result: irradiation control group irradiated cells apoptosis rate obviously increases (31% ± 1.19%vs, 14.9% ± 0.89%in control; P<0.01); and SDLF-501 group significantly can reduce irradiated cells apoptosis rate (18.5% ± 1.38%in model; P<0.01), SDLF-501 group is pointed out to have provide protection significantly (Fig. 5) to the cell injury that irradiation causes.
4. conclusion
According to above experimental result, SDLF-501 group can delay radiation damage survival time of animals significantly, extends its survival rate.SDLF-501 group has improvement result significantly to radiation damage marrow hemopoietic function.SDLF-501 group all improves in prolongation animal survival rate, hemopoietic function of bone marrow etc.
Embodiment 3 SDLF-501 animal acute toxicity test
According to preliminary experiment, drug toxicity is less, from 3g/kg, is incremented to 4g/kg and 5g/kg successively, tentatively determines 0% dead dosage and 100% dead dosage, determines medium lethal dose.
Result: give single oral gavage administration, at 3g/kg dosage without animal dead (n=10); At 4g/kg dosage without animal dead (n=10); At 5g/kg dosage without animal dead (n=10);
Conclusion: SDLF-501 is hypotoxicity material.
The preparation (one) of embodiment 4 SDLF-501 monocrystalline
Example 1 prepare refining after formula I product 1g, add methylene dichloride 30ml, normal hexane 10ml, return time 5 hours, clarification, places 20 hours, slowly crystallization, obtains colourless granules shape single crystal compound.
The single crystal analysis of embodiment 5 X-radiocrystallography
1. experimental technique
At 223K temperature, be equipped with graphite-monochromatization Mo K α radiation
bRUKER SMART CCD diffractometer on collect the crystal data of SDLF-501 monocrystalline prepared by embodiment 4.Whole data set is used to measure final unit cell parameters.
2. experimental result
The crystal data of SDLF-501 monocrystalline is in table 1, and hierarchical atoms coordinate is in table 2.Those skilled in the art should know, and the slight change of coordinate is possible, and are considered within open scope of the present invention.
The crystal data of table 1 SDLF-501 monocrystalline
In above-mentioned table 1, crystal data translation is as follows, when Chinese and English exists inconsistent, is as the criterion with the English content that table 1 is recorded.
The atomic coordinate (× 10 of table 2 SDLF-501 monocrystalline
4) and equivalent isotropic displacement parameter
u (eq) is defined as orthogonalization U
ij/ 3rd of a tensor trace
The powder x-ray diffraction of embodiment 6 SDLF-501 monocrystalline
1. experimental technique
Use Bruker D8Advance x-ray powder diffraction instrument to obtain powder x-ray diffraction, radiation is Cu target.
2. experimental result
The powder x-ray diffraction spectrogram of SDLF-501 monocrystalline is shown in Fig. 6, and Fig. 7 is concrete each peak position data, the Y-axis in Height and Fig. 6 in Fig. 7, is counting.There is peak the diffraction angle of 13.5 °, 15.2 °, 20.5 °, 21.8 °, 22.8 °, 25.5 °, 27.9 ° in SDLF-501 monocrystalline, concrete outcome is as the criterion with Fig. 6 and Fig. 7.
The preparation (two) of embodiment 7 SDLF-501 monocrystalline
Example 1 prepare refining after formula I product 1g, add methylene dichloride 10ml, normal hexane 70ml, return time 1 hour, clarification, place 30 hours, slowly crystallization, obtains single crystal compound.
The powder x-ray diffraction spectrum of this monocrystalline is consistent with the monocrystalline that embodiment 4 obtains.
The preparation (three) of embodiment 8 SDLF-501 monocrystalline
Example 1 prepare refining after formula I product 1g, add methylene dichloride 80ml, normal hexane 5ml, return time 10 hours, clarification, place 1 hour, slowly crystallization, obtains single crystal compound.
The powder x-ray diffraction spectrum of this monocrystalline is consistent with the monocrystalline that embodiment 4 obtains.
The preparation (four) of embodiment 9 SDLF-501 monocrystalline
Example 1 prepare refining after formula I product 1g, add methylene dichloride 50ml, normal hexane 60ml, return time 8 hours, clarification, place 5 hours, slowly crystallization, obtains single crystal compound.
The powder x-ray diffraction spectrum of this monocrystalline is consistent with the monocrystalline that embodiment 4 obtains.
The stability experiment of embodiment 10 SDLF-501 monocrystalline
1 experimental technique
SDLF-501 monocrystalline is placed on lower 15 days of 30 DEG C of conditions by normal temperature condition, respectively at the 5th, 10,15 day sampling and measuring powder x-ray diffraction spectrogram, judges the stability of SDLF-501 monocrystalline.
2 experimental results
Under 30 DEG C of conditions 15 days, SDLF-501 monocrystalline was stablized, and outward appearance remains unchanged, and considerable change does not occur powder x-ray diffraction spectrogram.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (10)
1. prevent and treat a compound for radiation injury, it is characterized in that, the chemical structure of described compound is such as formula shown in I:
2. a preparation method for formula I, is characterized in that, comprises the following steps:
A) 3-(6 is got, 7-dihydro-1-methyl-7-oxo-3-propyl group-1H-pyrazoles [4,3-d] pyrimidine-5-yl)-4-oxyethyl group-benzene sulfonyl, be dissolved in chloroform, ice bath, stirring and dissolving, add triethylamine, 4-methylimidazole, stir, TCL monitors reaction, run plate and lose main raw material, stopped reaction;
B) reaction solution adds saturated sodium bicarbonate solution washing, then with sodium chloride solution washing, separates organic phase, dry;
C) filtrate evaporate to dryness, obtains oily matter, adds isopropyl ether, acetonitrile, and clarification, stirring at room temperature, slowly separates out white solid, obtains described formula I.
3. the monocrystalline of a formula I, it is characterized in that, there is characteristic peak at the following 2 θ angles of described monocrystalline in powder x-ray diffraction collection of illustrative plates: 13.5 ± 0.1 °, 15.2 ± 0.1 °, 20.5 ± 0.1 °, 21.8 ± 0.1 °, 22.8 ± 0.1 °, 25.5 ± 0.1 °, 27.9 ± 0.1 °.
4. a monocrystalline for formula I, is characterized in that, described monocrystalline characterizes as follows:
Crystallographic system: triclinic(crystalline)system
Spacer: P-1
Unit cell dimension:
α＝88.330(4)°
β＝77.426(4)°
γ＝79.072(4)°
Volume:
Z＝2。
5. the preparation method of the monocrystalline described in claim 3 or 4, is characterized in that, comprises the following steps: modus ponens I, adds methylene dichloride and normal hexane, backflow, clarification, and place, slowly crystallization, obtains monocrystalline.
6. preparation method according to claim 5, is characterized in that, comprises the following steps: modus ponens I, adds methylene dichloride and normal hexane, backflow 1-10 hour, and clarification, places 1-30 hour, slowly crystallization, obtain monocrystalline; Described methylene dichloride and formula I ratio are 10-80ml:1g, and described normal hexane and formula I ratio are 5-70ml:1g.
7. the pharmacy acceptable salt of compound according to claim 1 or the monocrystalline described in claim 3 or 4, it is characterized in that, described salt is inorganic acid salt or organic acid salt, and described inorganic acid salt is hydrochloride, vitriol, phosphoric acid salt, diphosphate, hydrobromate or nitrate; Described organic acid salt is acetate, maleate, fumarate, tartrate, succinate, lactic acid salt, tosilate, salicylate or oxalate.
8. a pharmaceutical composition, is characterized in that, described pharmaceutical composition contains the treatment compound according to claim 1 of significant quantity, the monocrystalline described in claim 3 or 4 or salt according to claim 7, and the pharmaceutically acceptable carrier of surplus.
9. the pharmaceutical applications of the monocrystalline described in compound according to claim 1, claim 3 or 4 or salt according to claim 7, is characterized in that, for the preparation of the medicine of control radiation injury.
10. the pharmaceutical applications of the monocrystalline described in compound according to claim 1, claim 3 or 4 or salt according to claim 7, is characterized in that, for:
A) red corpuscle suppressing radiation to cause and leukocyte count object reduce,
B) red corpuscle suppressing radiation to cause and the reduction of platelet content,
C) alleviating of the spleen weight preventing radiation from causing,
D) medullary cell is improved to the tolerance of radiation, or
E) lost of life suppressing radiation to cause.
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Citations (4)
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|---|---|---|---|---|
| US3892824A (en) * | 1968-12-16 | 1975-07-01 | Southern Res Inst | S-{107 -({107 -aminoalkylamino)alkyl dihydrogen phosphorothioates |
| CN1333694A (en) * | 1998-11-16 | 2002-01-30 | 美国生物科学有限公司 | Stable amorphous amifostine compositions and methods for the preparation and use of same |
| CN1312153C (en) * | 2000-02-17 | 2007-04-25 | Sk化学株式会社 | Pyrrolopyrimidinone derivatives, process of preparation and use |
| CN103816531A (en) * | 2013-07-09 | 2014-05-28 | 许颖 | Application of fusion protein in preparing drug, health food or food for curing and/or preventing radiation damage |
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2015
- 2015-03-20 CN CN201510125429.8A patent/CN104744478B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3892824A (en) * | 1968-12-16 | 1975-07-01 | Southern Res Inst | S-{107 -({107 -aminoalkylamino)alkyl dihydrogen phosphorothioates |
| CN1333694A (en) * | 1998-11-16 | 2002-01-30 | 美国生物科学有限公司 | Stable amorphous amifostine compositions and methods for the preparation and use of same |
| CN1312153C (en) * | 2000-02-17 | 2007-04-25 | Sk化学株式会社 | Pyrrolopyrimidinone derivatives, process of preparation and use |
| CN103816531A (en) * | 2013-07-09 | 2014-05-28 | 许颖 | Application of fusion protein in preparing drug, health food or food for curing and/or preventing radiation damage |
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