CN104876979B - One kind having active sulfonated five sugar compounds of Anti-Xa factor - Google Patents
One kind having active sulfonated five sugar compounds of Anti-Xa factor Download PDFInfo
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Abstract
It is the derivative of five sugar compounds with blood coagulation resisting function to have active sulfonated five sugar compounds of Anti-Xa factor and its preparation method and application, this compound the present invention relates to one kind, has significant Anti-Xa factor activity.
Description
Technical field
The invention belongs to pharmaceutical technology fields, are related to a kind of compound, and in particular to one kind has Anti-Xa factor active
Sulfonated five sugar compounds.
Technical background
Thrombotic diseases are the diseases for seriously endangering human health, according to thrombosis position, condition and property, main point
For arterial thrombus and phlebothrombosis.Arterial thrombosis is from arterial blood tube wall atherosclerotic lesion and platelet activation
Start, caused bad clinical disease is mainly acute myocardial infarction AMI, cerebral apoplexy；Phlebothrombosis is by a variety of in vein blood vessel
Reason induces to be formed, and it is Deep vain thrombosis and pulmonary embolism that can lead to venous thromboembolism, main clinical manifestation.Big rule
Mould clinical test evidence shows that anticoagulant therapy can prevent the sprawling and recurrence of thrombus, and further decrease cerebral apoplexy, pulmonary embolism
Deng incidence and the death rate.Therefore, anticoagulant therapy has become current clinical prevention and treats the core of thrombotic disease
And basis, and the research and development of anticoagulant are also the hot spot of new drug development always.
Currently, the novel anticoagulant researched and developed or listed includes mainly direct thrombin inhibitor, Xa factor
Inhibitor, IX factor inhibitors, tissue factor inhibitor and novel vitamin K antagon.Wherein, direct fibrin ferment inhibits
Agent and Xa factor inhibitors are most representative anti-freezing new drugs.
Fondaparinux sodium is a kind of artificial synthesized Selective activation factor (Xa) inhibitor, passes through the selection with AT III
Property combine, enhance the AT III neutralizations intrinsic to factor Xa, the neutralization of factor Xa may interfere with coagulation cascade system
System, and fibrin ferment can be inhibited to be formed simultaneously and (indicated respectively from a left side with D, E, F, G, H as follows with thrombus development, chemical structural formula
To right 5 monosaccharide)：
Invention content
Present invention aims at provide one kind there are active sulfonated five sugar compounds of Anti-Xa factor (hereinafter referred to as to change
Close object A).
Compound A of the present invention, it is 612 with extraordinary Anti-Xa factor activity, anti-Xa potency after measured
IU/mg is approximately 1.5 times of Fondaparinux sodium, has high medical value and significant therapeutic effect.
Compound A of the present invention, chemical structural formula (indicate 5 from left to right with D, E, F, G, H respectively as follows
Monosaccharide)：
It is another object of the present invention to provide the preparation methods of compound A.
Specifically, the synthetic route of the compound of the present invention A is as follows：
The preparation method of compound A of the present invention, includes the following steps：
Step 1, the preparation of D：
D1 is dissolved with appropriate anhydrous methylene chloride, Tritox is added, DBU is added, 0.5h is reacted at room temperature, obtains product D；
Step 2, the preparation of DEF：
, D and EF are dissolved in appropriate anhydrous methylene chloride, appropriate dry 4A molecular sieves are added, stir 30 minutes at room temperature
Afterwards, the dichloromethane solution of Trimethylsilyl trifluoromethanesulfonate is added at nitrogen protection, -20 DEG C, reacts 30 minutes, obtains
DEF1.DEF1 is dissolved in appropriate acetic anhydride, the dichloromethane of Trimethylsilyl trifluoromethanesulfonate is added at nitrogen protection, 0 DEG C
Alkane solution reacts at room temperature 3 hours；Ammonium hydroxide is added, is reacted at room temperature 4 hours；It is eventually adding appropriate Tritox and DBU, nitrogen
Gas shielded reacts at room temperature 3 hours, obtains product DEF；
Step 3, the preparation of GH：
, G, BSP and 4A molecular sieve are dissolved in anhydrous methylene chloride, 30min is stirred at room temperature under nitrogen protection, is cooled to -60
DEG C, Tf is slowly added dropwise2O, after drop finishes, temperature rises to -40 DEG C, and the H after using anhydrous methylene chloride dissolved dilution is added, and -40 DEG C anti-
1h is answered, product G H1 is obtained.GH1 is dissolved in appropriate anhydrous methylene chloride, triethylamine is added at room temperature, 3h is reacted, obtains product
GH；
Step 4, the preparation of DEFGH：
, DEF and GH are dissolved in appropriate anhydrous methylene chloride, appropriate dry 4A molecular sieves are added, are stirred at room temperature 30 minutes
Afterwards, the dichloromethane solution of appropriate Trimethylsilyl trifluoromethanesulfonate is added at nitrogen protection, -20 DEG C, reacts 1 hour, adds
Enter appropriate triethylamine, stir 30 minutes, filtering, filtrate is concentrated to dryness, and q. s. methylene chloride dissolving is added, is washed with water successively, nothing
Aqueous sodium persulfate is dried, and filtering, filtrate is concentrated to dryness, and is crossed silicagel column, is obtained product DEFGH；
Step 5, the preparation of compound A
, DEFGH is dissolved in THF, purified water is added, NaOH solution is added dropwise later, is reacted 18 hours, hydrochloric acid is added dropwise and neutralizes,
Then chloroform extraction is added, takes organic phase, is concentrated to dryness.Gained sample is dissolved in DMF, and sulfur trioxide front three is added
Amine complex, nitrogen protection are reacted 18 hours, are concentrated under reduced pressure, filtrate loading to sephadex lh-20 chromatographic column desalination, will
The product liquid of collection is evaporated under reduced pressure, and loading is to 732 sodium form storng-acid cation exchange resin chromatographic columns, by the product of collection
Liquid is concentrated to dryness.Gained sample, purified water and 10% palladium carbon are added in hydriding reactor, in kettle plus hydrogen is to 1.5MPa, reacts
68 hours, after reaction, filtering concentrated filtrate decompression.Condensate residue is moved in reaction kettle, purified water is added, it will
Sulfur trioxide pyridine complex is added portionwise in reaction kettle, reacts 7 hours, until after solution clear, filtering, loading to Portugal
The product liquid of collection is evaporated under reduced pressure by polysaccharide gel G-25 chromatographic column desalinations, and loading to 732 sodium form highly acidic cations are handed over
Resin chromatography column is changed, the product liquid of collection is concentrated to dryness, compound A is obtained after purification through preparing.
Preferably, the preparation method of the compounds of this invention A, includes the following steps：
Step 1, the preparation of D：
D1 (18.0g, 42.1mmol) is dissolved in 90ml anhydrous methylene chlorides, addition Tritox (21ml,
209.4mmol), DBU (1.3ml, 8.7mmol) is added, reacts at room temperature 0.5h.It is concentrated to dryness, crosses silicagel column, obtain product D
(22.6g, 39.6mmol).
Step 2, the preparation of DEF：
D (22.6g, 39.6mmol) and EF (17.5g, 29.2mmol) are dissolved in 200ml anhydrous methylene chlorides, are added
40g dry 4A molecular sieves, at room temperature stir 30 minutes after, at nitrogen protection, -20 DEG C be added TBSOTf (2.7ml,
Dichloromethane solution 11.8mmol) reacts 30 minutes, crosses silicagel column, obtains DEF1 (19.0g, 18.8mmol).By DEF1
(19.0g, 18.8mmol) is dissolved in 95ml acetic anhydrides, is added TBSOTf's (0.9ml, 3.9mmol) at nitrogen protection, 0 DEG C
Dichloromethane solution reacts at room temperature 3 hours；Ammonium hydroxide 2ml is added, is reacted at room temperature 4 hours；It is eventually adding Tritox
(11.3ml, 113.0mmol) and DBU (0.4ml, 2.7mmol), nitrogen protection react at room temperature 3 hours, cross silicagel column, must produce
Product DEF (11.1g, 9.2mmol).
Step 3, the preparation of GH：
G (30.0g, 41.8mmol), BSP (11.4g, 54.6mmol) and 30g the 4A molecular sieves dried are dissolved in 150ml
Anhydrous methylene chloride is stirred at room temperature 30min under nitrogen protection, is cooled to -60 DEG C, Tf is slowly added dropwise2O (9.6ml,
58.5mmol), after drop finishes, temperature rises to -40 DEG C, be added with after anhydrous methylene chloride dissolved dilution H (23.1g,
50.4mmol), -40 DEG C of reaction 1h, cross silicagel column, obtain product G H1 (16.5g, 15.8mmol).By GH1 (16.5g,
It 15.8mmol) is dissolved in 150ml anhydrous methylene chlorides, triethylamine (55ml, 294.7mmol) is added at room temperature, react 3h,
Silicagel column is crossed, product G H (11.8g, 14.0mmol) is obtained.
Step 4, the preparation of DEFGH：
DEF (11.1g, 9.2mmol) and GH (9.3g, 11.0mmol) are dissolved in 100ml anhydrous methylene chlorides, are added
20g drying 4A molecular sieves, after being stirred at room temperature 30 minutes, at nitrogen protection, -20 DEG C be added TMSOTf (0.7ml,
Dichloromethane solution 3.9mmol) reacts 1 hour, and appropriate triethylamine is added, and stirs 30 minutes, and filtering, filtrate is concentrated into
It is dry, q. s. methylene chloride dissolving is added, is washed with water successively, anhydrous sodium sulfate drying is filtered, and filtrate is concentrated to dryness, and crosses silica gel
Column obtains product DEFGH (12.2g, 6.4mmol).
Step 5, the preparation of compound A：
DEFGH (12.2g, 6.4mmol) is dissolved in 150ml tetrahydrofurans, 80ml purified waters are added, 1N is added dropwise later
Sodium hydroxide solution 120ml is reacted at room temperature 18 hours, and 1N hydrochloric acid is added dropwise and neutralizes, chloroform extraction is then added, takes organic
Phase is concentrated to dryness.Gained sample is dissolved in 100ml n,N-Dimethylformamide, and sulfur trioxide trimethylamine complexing is added
Object (15.5g, 112.0mmol), nitrogen protection, 60 DEG C are reacted 18 hours, are concentrated under reduced pressure, filtrate loading to sephadex LH-
The product liquid of collection is evaporated under reduced pressure by 20 chromatographic column desalinations, and loading to 732 sodium form storng-acid cation exchange resins chromatograph
The product liquid of collection is concentrated to dryness by column.Gained sample, 360ml purified waters and 10% palladium carbons of 10.8g are added and hydrogenated
In kettle, in kettle plus hydrogen is to 1.5MPa, and 30 DEG C are reacted 68 hours, and after reaction, filtering concentrates filtrate decompression.Concentration is surplus
Excess moves in reaction kettle, and purified water is added, reaction is added portionwise in sulfur trioxide pyridine complex (9.5g, 59.7mmol)
In kettle, 10 DEG C are reacted 7 hours, until after solution clear, filtering, loading will be received to -25 chromatographic column desalination of sephadex G
The product liquid of collection is evaporated under reduced pressure, and loading is to 732 sodium form storng-acid cation exchange resin chromatographic columns, by the product liquid of collection
It is concentrated to dryness, compound A (2.6g, 1.5mmol) is obtained after purification through preparing.
It is another object of the present invention to provide a kind of pharmaceutical compositions containing compound A.
Pharmaceutically active substance in the pharmaceutical composition of the present invention is the compound of the present invention, in the formulation shared weight
Percentage can be 0.01-99.99%, remaining is pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention, can be prepared into any pharmaceutical dosage form as needed when in use, such as oral
Dosage form, injection form etc..
Pharmaceutically acceptable load can be added when being prepared into pharmaceutical preparation in the pharmaceutical composition of the present invention as needed
Body.
The pharmaceutical composition of the present invention exists with the dosage form of unit dose, and the unit dose refers to the list of preparation
Position, such as every of tablet, every capsule of capsule, every bottle of oral solution, every bag of granule, every etc. of injection.
The pharmaceutical composition of the present invention can be any pharmaceutical dosage form, these dosage forms include：Tablet, sugar coated tablet,
Film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral solution, mouth containing agent, granule, electuary, ball
Agent, powder, paste, sublimed preparation, suspension, pulvis, solution, injection, suppository, ointment, emplastrum, creme, spray,
Drops, pill, patch.
The preparation of the pharmaceutical composition of the present invention, oral medication can contain common excipient, such as adhesive, filling
Agent, diluent, tablet agent, lubricant, disintegrant, colorant, flavoring agent and wetting agent when necessary can be coated tablet.
Applicable filler includes cellulose, mannitol, lactose and other similar fillers.Suitable disintegrant packet
Include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant includes, such as firmly
Fatty acid magnesium.Suitable pharmaceutically acceptable wetting agent includes lauryl sodium sulfate.
The present invention can be by mixing, and filling, commonly method prepares solid oral composition for tabletting etc..It is mixed repeatedly
Active material can be made to be distributed in entirely to use in those of a large amount of fillers composition.
The form of oral liquid for example can be aqueous or oily suspensions, solution, emulsion, syrup or elixir,
Or can be a kind of dry products that water or other suitable carrier compoundings can be used before use.This liquid preparation can contain
There are conventional additive, such as suspending agent, such as sorbierite, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxylic first
Base cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifier, such as lecithin, anhydro sorbitol monooleate or Ah
Draw primary glue；Non-aqueous carrier (they may include edible oil), for example, the ester of apricot kernel oil, fractionated coconut oil, such as glycerine oil
Property ester, propylene glycol or ethyl alcohol；Preservative, such as para hydroxybenzene methyl esters or propylparaben or sorbic acid, and if
It needs, contains conventional flavouring agent or colorant.
For injection, the fluid unit dosage form of preparation contains the active material and sterile carrier of the present invention.According to carrier
And concentration, this compound can be suspended or be dissolved.The preparation of solution is typically by the way that active material is dissolved in a kind of load
In body, disinfection is filtered before being loaded into a kind of suitable bottle or ampoule, is then sealed.For example a kind of local anaesthesia of auxiliary material
Agent, preservative and buffer can also be dissolved in this carrier.In order to improve its stability, can be incited somebody to action after being packed into bottle
This composition frost, and under vacuum remove water.
The pharmaceutical composition of the present invention, suitable pharmaceutically acceptable load is optionally added when being prepared into medicament
Body, the pharmaceutically acceptable carrier are selected from：Mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, sodium thiosulfate,
Cysteine hydrochloride, thioacetic acid, methionine, injection Vitamin B_6 DTA disodiums, Ethylenediaminetetraacetic Acid Calcium Salt, the carbonate of monovalence alkali metal,
Acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylose
Alcohol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, fiber
Element and its derivative, alginates, gelatin, polyvinylpyrrolidone, glycerine, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, table
Face activating agent, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid material, kaolin, talcum powder, calcium stearate, stearic acid
Magnesium etc..
Preferably, pharmaceutical dosage form of the present invention is injection.
Injection of the present invention, the content of compound A is 1.5mg-6.5mg in unit formulation.
It is another object of the present invention to provide the medicinal usages of compound A.
Compound A of the present invention is preparing the application in having the active drug of Anti-Xa factor.
Applications of the compound A of the present invention in the drug for preparing treatment thrombotic diseases.
Thrombotic diseases of the present invention are broadly divided into arterial thrombus and Venous Thrombosis.
The present invention also provides the assay methods of compound A chemical constitutions and purity.
The method for detecting purity of compound A of the present invention, includes the following steps：
Using high performance liquid chromatograph, using quaternary ammonium anion shell resin as the polymeric anion exchange column of filler, water
It is mobile phase, the purity of detection compound A with sodium chloride solution.
The specific detection method of the present invention is see specification embodiment 2.
The present invention also provides the structure determinations of compound A.
The structure that the present invention passes through nuclear-magnetism and Mass Spectrometric Identification compound A.In terms of nucleus magnetic hydrogen spectrum, compared with Fondaparinux sodium,
More a methyl peak, the hydrogen chemical shifts of H sugar 2 shift to low field 3.66 by 3.25 at 2.74, and the hydrogen of H sugar 3 is moved by 3.62
To 3.93, other positions variation is little.It is composed from nuclear-magnetism carbon, compared with Fondaparinux sodium, more carbon peaks, H at 32.76
The carbon that the carbon of sugar 2 shifts to 62.85, H sugar 1 and 3 by 60.37 also has different degrees of variation, other positions to change not
Greatly.It is detected by high resolution mass spectrum, infers that the accurate molecular weight of the compound is：1740.7864, molecular formula is
C32H45N3Na10O49S8.It may infer that in conjunction with mass spectrum and replaced by methyl with the hydrogen on sugared 2 amino being connected of H so that H sugar 2
The chemical shift of hydrogen shift to low field, meanwhile, H sugar 1 is also changed with 3.
The present invention promulgates according to State Food and Drug Administration, the national drug standards of low molecular weight calcium heparin
Determination method measures Anti-Xa factor (FXa) activity of compound A in WS1- (X-147) -2005Z.Measure the anti-of compound A
Xa factor potency is 612IU/mg, and Fondaparinux sodium potency is 400IU/mg, and low molecular weight calcium heparin potency is 105IU/mg.
Compound A belongs to new compound, and domestic and foreign literature patent does not report it at present.By to compound A
Anti-Xa factor activity be measured, anti-Xa potency is 612IU/mg, and about the 1.5 of Fondaparinux sodium times are shown
Better Anti-Xa factor activity, future are likely to become upgrading or the Regeneration variety of Fondaparinux sodium.
In addition, as new compound, in terms of compound A may also have the function of other potential pharmacology, from long-range
Consider, by more in-depth study, also has higher value.
The compound of the present invention A is compared with existing product compared with having bioactivity strong, the features such as few side effects.Such as compare sulphur
There is stronger bioactivity up to liver last of the ten Heavenly stems sodium and Low-molecular-weight Heparins Calcium；Compound A belongs to single compound, than belonging to mixture
Low-molecular-weight Heparins Calcium few side effects.
Compound D1, EF, G of the present invention, H, belong to existing product, can buy on the market.
Noun described herein is further explained：
DBU：11 carbon -7- alkene of 1,8- diazabicylos
TBSOTf：Trimethylsilyl trifluoromethanesulfonate
TMSOTf：Trifluoromethanesulfonic acid trimethyl silicone grease
BSP：1- (phenylsulfmyl) piperidines
Above-mentioned raw materials belong to existing product, can be commercially available.
Description of the drawings
Fig. 1：The liquid chromatogram of the compounds of this invention A
Specific implementation mode
The present invention is further explained by following specific examples, but not as the limitation of the present invention.
Embodiment 1：The preparation of compound A
Step 1, the preparation of D：
D1 (18.0g, 42.1mmol) is dissolved in 90ml anhydrous methylene chlorides, addition Tritox (21ml,
209.4mmol), DBU (1.3ml, 8.7mmol) is added, reacts at room temperature 0.5h.It is concentrated to dryness, crosses silicagel column, obtain product D
(22.6g, 39.6mmol).
Step 2, the preparation of DEF：
D (22.6g, 39.6mmol) and EF (17.5g, 29.2mmol) are dissolved in 200ml anhydrous methylene chlorides, are added
40g dry 4A molecular sieves, at room temperature stir 30 minutes after, at nitrogen protection, -20 DEG C be added TBSOTf (2.7ml,
Dichloromethane solution 11.8mmol) reacts 30 minutes, crosses silicagel column, obtains DEF1 (19.0g, 18.8mmol).By DEF1
(19.0g, 18.8mmol) is dissolved in 95ml acetic anhydrides, is added TBSOTf's (0.9ml, 3.9mmol) at nitrogen protection, 0 DEG C
Dichloromethane solution reacts at room temperature 3 hours；Ammonium hydroxide 2ml is added, is reacted at room temperature 4 hours；It is eventually adding Tritox
(11.3ml, 113.0mmol) and DBU (0.4ml, 2.7mmol), nitrogen protection react at room temperature 3 hours, cross silicagel column, must produce
Product DEF (11.1g, 9.2mmol).
Step 3, the preparation of GH：
G (30.0g, 41.8mmol), BSP (11.4g, 54.6mmol) and 30g the 4A molecular sieves dried are dissolved in 150ml
Anhydrous methylene chloride is stirred at room temperature 30min under nitrogen protection, is cooled to -60 DEG C, Tf is slowly added dropwise2O (9.6ml,
58.5mmol), after drop finishes, temperature rises to -40 DEG C, be added with after anhydrous methylene chloride dissolved dilution H (23.1g,
50.4mmol), -40 DEG C of reaction 1h, cross silicagel column, obtain product G H1 (16.5g, 15.8mmol).By GH1 (16.5g,
It 15.8mmol) is dissolved in 150ml anhydrous methylene chlorides, triethylamine (55ml, 294.7mmol) is added at room temperature, react 3h,
Silicagel column is crossed, product G H (11.8g, 14.0mmol) is obtained.
Step 4, the preparation of DEFGH：
DEF (11.1g, 9.2mmol) and GH (9.3g, 11.0mmol) are dissolved in 100ml anhydrous methylene chlorides, are added
20g drying 4A molecular sieves, after being stirred at room temperature 30 minutes, at nitrogen protection, -20 DEG C be added TMSOTf (0.7ml,
Dichloromethane solution 3.9mmol), react 1 hour, be concentrated to dryness, cross silicagel column, obtain product DEFGH (12.2g,
6.4mmol)。
Step 5, the preparation of compound A：
DEFGH (12.2g, 6.4mmol) is dissolved in 150ml tetrahydrofurans, 80ml purified waters are added, 1N is added dropwise later
Sodium hydroxide solution 120ml reacts 18 hours, and 1N hydrochloric acid is added dropwise and neutralizes, chloroform extraction is then added, takes organic phase, subtracts
Pressure is concentrated to dryness.Gained sample is dissolved in 100ml n,N-Dimethylformamide, and sulfur trioxide trimethylamine complex compound is added
(15.5g, 112.0mmol), nitrogen protection are reacted 18 hours, are concentrated under reduced pressure, and filtrate loading to sephadex lh-20 chromatographs
The product liquid of collection is evaporated under reduced pressure by column desalination, and loading will be received to 732 sodium form storng-acid cation exchange resin chromatographic columns
The product liquid of collection is concentrated to dryness.Gained sample, 360ml purified waters and 10% palladium carbons of 10.8g are added in hydriding reactor, kettle
Interior plus hydrogen reacts 68 hours, after reaction, filtering concentrates filtrate decompression to 1.5MPa.Condensate residue is moved to instead
It answers in kettle, purified water is added, sulfur trioxide pyridine complex (9.5g, 59.7mmol) is added portionwise in reaction kettle, reaction 7
Hour, until after solution clear, filtering, loading to -25 chromatographic column desalination of sephadex G depressurizes the product liquid of collection
Distillation, and the product liquid of collection is concentrated to dryness by loading to 732 sodium form storng-acid cation exchange resin chromatographic columns, warp
It prepares and obtains compound A (2.6g, 1.5mmol) after purification.
Embodiment 2 passes through high performance liquid chromatograph device detection compound A
Chromatographic column is Dionex CarboPacTMPA1 (250 × 4mm), flow velocity 1.0ml/min, Detection wavelength are
210nm, column temperature is 35 DEG C, using water as mobile phase A (about 10 μ l dimethyl sulfoxides are added in 1000ml water), 116.9g/l sodium chloride
Solution is Mobile phase B, and it is as shown in the table for gradient：
The purity detecting result of compound A is as shown in Fig. 1：Retention time is 16.304min, purity 99.73%.
Embodiment 3：Structural Identification is carried out to compound A by nuclear-magnetism and mass spectrum
Instrument testing conditions：6520 Q-TOF LCMS high-resolution mass spectrometers of Agilent, flow velocity 1ml/min, 1 μ l of sample introduction,
Ion source ESI；BRUKER 400MHz Nuclear Magnetic Resonance；Solvent：D2O；Internal standard：TMS.Detection data is as follows：
1 hydrogen of table is composed and carbon modal data
2 high resolution mass spectrum data of table
In terms of nucleus magnetic hydrogen spectrum, as shown in table 1, compared with Fondaparinux sodium, at 2.74 more methyl peaks, H sugar 2
Hydrogen chemical shifts shift to low field 3.66 by 3.25, and the hydrogen of H sugar 3 shifts to 3.93 by 3.62, and other positions variation is little.From core
Magnetic carbon spectrum is seen, compared with Fondaparinux sodium, more a carbon peak, the carbon of H sugar 2 shift to 62.85, H sugar by 60.37 at 32.76
1 and 3 carbon also has different degrees of variation, other positions variation little.It is detected by high resolution mass spectrum, such as 2 institute of table
Show, infers that the accurate molecular weight of the compound is：1740.7864 molecular formula C32H45N3Na10O49S8.It can be in conjunction with mass spectrum
Infer and replaced by methyl with the hydrogen on sugared 2 amino being connected of H so that low field is shifted in the chemical shift of the hydrogen of H sugar 2, together
When, H sugar 1 is also changed with 3.It thereby determines that, the structural formula of compound A is as follows：
Embodiment 4：Xa factor (FXa) Activity determination of compound A
It is promulgated according to State Food and Drug Administration, the national drug standards WS1- (X- of low molecular weight calcium heparin
147) Determination method measures in -2005Z, i.e., is marked in vitro by Antithrombin III (AT III) and low molecular weight heparin (LMWH)
Quasi- product compare to measure the activity that test sample accelerates inhibition Xa factor (FXa).The specific method is as follows：
One, solution is prepared
Trishydroxymethylaminomethane-sodium chloride buffer (pH 7.4)：Take trishydroxymethylaminomethane 6.08g and sodium chloride
8.77g adds water 500ml to make dissolving, adds bovine serum albumin(BSA) 10g, with salt acid for adjusting pH value to 7.4, is diluted with water to 1000ml.
Trishydroxymethylaminomethane-Calcium Disodium Versenate buffer solution (pH 8.4)：Take sodium chloride 5.12g, three hydroxyl first
Base aminomethane 3.03g and Calcium Disodium Versenate 1.4g, adds water 250ml to make dissolving, with salt acid for adjusting pH value to 8.4, adds
Water is diluted to 500ml.
The preparation of LMWH standard items and test solution：With trishydroxymethylaminomethane-sodium chloride buffer (pH 7.4)
Standard items (S) and test sample (T) are diluted to the solution of 4 various concentrations respectively, the agent between each dosage is away from than being generally 1:
0.7~1:0.6 concentration should be in the range of linearity of log dose-responses.Contain 0.025IU~0.2IU in generally every 1ml.
Antithrombase (AT III) solution：Every 1ml is made with trishydroxymethylaminomethane-sodium chloride buffer (pH 7.4)
In the solution containing 1IU.
Chromophoric substrate s-2765 (or other FXa specificity chromophoric substrates)：The solution of 0.003mol/L is made of water for solution,
Before use 0.0005mol/L is diluted to trishydroxymethylaminomethane-Calcium Disodium Versenate buffer solution (pH 8.4).
Xa factor (FXa) solution：With trishydroxymethylaminomethane-sodium chloride buffer (pH 7.4), FXa solution is prepared,
Its concentration is debugged, is allowed in the Anti-Xa factor experiment for replacing LMWH with 0.9% sodium chloride solution, at the wavelength of 405nm
Absorption value is 0.6~0.7.
Two, measuring method
Small test tube 16 is taken, marks T respectively1、T2、T3、T4And S1、S2、S3、S4.Each concentration is parallel to do two pipes.Often pipe is distinguished
The test sample (T) or 50 μ l of standard items (S) dilution and 50 μ l of antithrombase solution, mixing for adding 4 kinds of concentration of people (must not generate
Bubble).By S1、S2、S3、S4、T1、T2、T3、T4、T1、T2、T3、T4、S1、S2、S3、S4It is ranked sequentially, 37 DEG C balance 1 minute.Often
Pipe plus 100 μ l of FXa solution, mixing, 37 DEG C balance 1 minute, add 250 μ l of chromophoric substrate solution, mix, 37 DEG C of accurate 4 points of heat preservations
Zhong Hou, respectively plus 30% acetum, 375 μ l, termination are reacted.With the semimicro colorimetric pool of 1cm light paths, with trihydroxy methyl amino first
Alkane-sodium chloride buffer (pH 7.4) is blank, and the absorbance of often pipe is measured at the wavelength of 405nm.With three light methylaminos
Methane-sodium chloride buffer (pH 7.4) replaces test solution (parallel to do two pipes) to be used as blank control pipe with method operation,
When 16 branch pipe beginning and end, the absorbance of blank control pipe is measured respectively.The absorbance of the two must not significant difference.
Using absorbance as ordinate, the logarithm of standard solution (or test solution) concentration is that abscissa makees linear regression respectively,
By 4 × 4 method of the parallel line analysis principle experiment in Bioassay-statistical method (two annex X IV of China's coastal port)
Design calculates potency and experimental error.Average Reliable limit rate (FL%) is not greater than 15%.Three, test result
It is measured through the above method, the Anti-Xa factor potency of compound A is 612IU/mg, and Fondaparinux sodium potency is 400IU/
Mg, low molecular weight calcium heparin potency are 105IU/mg.Compared with Fondaparinux sodium, the Anti-Xa factor potency of compound A is about it
1.5 again.
5 compound A injections of embodiment
Prescription：
Preparation process：
Step 1, aseptically, 80% water for injection of recipe quantity is taken, the dissolving of recipe quantity sodium chloride is added, with 0.1M hydrogen
Sodium oxide molybdena or hydrochloric acid solution adjust pH to 5.0-8.0；
Step 2, aseptically, recipe quantity compound A is taken to be added in above-mentioned solution, stirring makes dissolving；
Step 3, aseptically, water for injection is added into above-mentioned acquired solution to full dose；
Step 4, aseptically, 0.1% medical charcoal is added to above-mentioned acquired solution, heat preservation is stirred 30 minutes；
Step 5, aseptically, above-mentioned acquired solution is dispensed into glass ampule to get finished product.
Claims (4)
1. compound A is preparing the application in having the active drug of Anti-Xa factor, which is characterized in that Compound A structure is as follows：
2. applications of the compound A in the drug for preparing treatment thrombotic diseases, the thrombotic diseases are arterial thrombus or quiet
Arteries and veins thrombotic diseases, which is characterized in that Compound A structure is as follows：
3. a kind of composition for injection containing compound A, is characterized in that, the content of compound A is 1.5mg- in unit formulation
6.5mg。
4. pharmaceutical composition according to claim 3, which is characterized in that preparation method includes the following steps：
Step 1, aseptically, 80% water for injection of recipe quantity is taken, the dissolving of recipe quantity sodium chloride is added, with 0.1M hydroxides
Sodium or hydrochloric acid solution adjust pH to 5.0-8.0；
Step 2, aseptically, recipe quantity compound A is taken to be added in above-mentioned solution, stirring makes dissolving；
Step 3, aseptically, water for injection is added into above-mentioned acquired solution to full dose；
Step 4, aseptically, 0.1% medical charcoal is added to above-mentioned acquired solution, heat preservation is stirred 30 minutes；
Step 5, aseptically, above-mentioned acquired solution is dispensed into glass ampule to get finished product.
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| CN201510345307.XA CN104876979B (en) | 2015-06-19 | 2015-06-19 | One kind having active sulfonated five sugar compounds of Anti-Xa factor |
| PCT/CN2016/095464 WO2016202310A1 (en) | 2015-06-19 | 2016-08-16 | Sulphonated pentasaccharide compound having anti-xa factor activity |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989146A (en) * | 2004-03-04 | 2007-06-27 | 普罗吉恩工业有限公司 | Sulfated oligosaccharide derivatives |
| US20140336369A1 (en) * | 2011-06-28 | 2014-11-13 | Apicore Us Llc | Process for preparing heparinoids and intermediates useful in the synthesis thereof |
| CN104245718A (en) * | 2009-07-31 | 2014-12-24 | 可靠生物医药公司 | Process for preparing fondaparinux sodium and intermediates useful in the synthesis thereof |
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| ES2009284A6 (en) * | 1988-06-15 | 1989-09-16 | Miret Lab | A process for the obtainment of copolimers with sequestering and dispersing properties. |
| JP3169343B2 (en) * | 1997-03-14 | 2001-05-21 | サノフィ | Compositions containing a combination of aspirin and anti-Xa oligosaccharide and use of anti-Xa oligosaccharide optionally in combination with aspirin |
| CA2459562C (en) * | 2001-09-07 | 2013-05-07 | Alchemia Pty. Ltd. | Synthetic heparin pentasaccharides |
| WO2010029552A1 (en) * | 2008-09-10 | 2010-03-18 | Transpharma Medical Ltd. | Transdermal delivery of oligosaccharides |
| US20130262309A1 (en) * | 2012-04-02 | 2013-10-03 | Mpayme Ltd. | Method and System for Secure Mobile Payment |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989146A (en) * | 2004-03-04 | 2007-06-27 | 普罗吉恩工业有限公司 | Sulfated oligosaccharide derivatives |
| CN104245718A (en) * | 2009-07-31 | 2014-12-24 | 可靠生物医药公司 | Process for preparing fondaparinux sodium and intermediates useful in the synthesis thereof |
| US20140336369A1 (en) * | 2011-06-28 | 2014-11-13 | Apicore Us Llc | Process for preparing heparinoids and intermediates useful in the synthesis thereof |
Non-Patent Citations (1)
| Title |
|---|
| Total Synthesis of Anticoagulant Pentasaccharide Fondaparinux;Tiehai Li，等;《ChemMedChem》;20140411;第9卷(第5期);第1071-1080页 * |
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| WO2016202310A1 (en) | 2016-12-22 |
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