CN109134554A - Anticoagulant five saccharide compound and preparation method thereof and medical usage - Google Patents
Anticoagulant five saccharide compound and preparation method thereof and medical usage Download PDFInfo
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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Abstract
The present invention relates to anticoagulation medicines, and in particular to one kind five sugar compounds of synthesis and its salt, preparation method and its pharmaceutical applications.The present invention has synthesized a kind of five sugar compounds as shown ionic species shown in II or its acid or its salt, has compared with the stronger anticoagulin xa activity of Fondaparinux sodium and longer elimination half-life period.Five sugar compounds of the invention can be used for preparing the drug of prevention and treatment Yu blood coagulation disorder related disease, such as diseases such as Deep vain thrombosis, thrombophlebitis, the artery occlusion as caused by thrombosis or embolism, Postoperative Intravenous thrombosis or embolisms.
Description
Technical field
The present invention relates to five saccharide compounds for being used as anticoagulant, preparation method and its doctor in terms of anticoagulation
Medicinal way.
Background technique
Thrombotic diseases are a kind of diseases for seriously endangering human health, and disease incidence is in first of various diseases, in recent years
Carry out also cumulative gesture.It is broadly divided into arterial thrombus and phlebothrombosis.Phlebothrombosis is common in Deep venou, and clinical manifestation is blood
Caused embolism causes organ function to hinder after local pain swelling, distal end blood backflow obstacle and thrombus breaks loose that bolt is formed
Hinder.Arterial thrombosis is can lead to serious since arterial blood tube wall atherosclerotic lesion and platelet activation
Cardiovascular disease such as acute myocardial infarction AMI, cerebral apoplexy etc..Treatment method includes anticoagulant therapy, Antiplatelet therapy and thromboembolism treatment
Deng, and anticoagulant therapy is core and the basis of current clinical prevention and treatment thrombotic diseases.
Heparin is traditional anticoagulation medicine, is one of glycosaminoglycan family polysaccharide.What is clinically used includes general
Logical heparin and low molecular weight heparin.The conformation that heparin induces AT and being attached to the particular combination domain of antithrombase (AT) occurs
Variation, and then inhibit the activity of factor Xa.Studies have shown that Heparin-binding AT and inhibiting the minimal structure list of factor Xa
Member is unique five glycosylation sequence.
Up to the present, have more documents and disclose and synthesize to obtain with antithrombus formation and anticoagulation by full chemistry
Active five sugar compounds.Such as United States Patent (USP) US4818816, Carbohydrate Research 1987 (167): 67-75 report
The Fondaparinux sodium in road is the first generation synthetic analogues that five glycosylation sequences are combined based on anticoagulant heparin hemase, is belonged to indirectly solidifying
Blood factor Xa inhibitor.Fondaparinux sodium is about 1 hour in rat intracorporal half-life period, is about 17 small intracorporal half-life period in people
When.The product since U.S.'s listing, in multiple country's listings, were clinically used to treat and prevent Deep vein blood from 2002
Bolt Embolic events occur, recommended dose 2.5mg, once a day, subcutaneous administrations.Although Fondaparinux sodium clinical manifestation goes out
Color, but its synthesis difficulty is very big, production cost is high, to increase the financial burden of patient.
United States Patent (USP) US5378829, which is reported, replaces N- in Fondaparinux sodium structure with alkoxy grp and O- sulfonate group
Sulfonate group, hydroxyl group and a new class of pentasaccharides formed, such as idraparinux sodium (Idraparinux).Due to pentasaccharides list
Alkoxy grp is introduced in member and O- sulfonate group, preparation mode are greatly simplified.It is 9.2 small in rat Half-life in vivo
When (iv), in the intracorporal Increased Plasma Half-life of people to 120h, bleeding risk increases, and stops at phase III clinical trial.
WO9936428 is reported by replacing O- alkyl with alkylidene bridge and locking the configuration of G unit L- iduronic acid
And five sugar compounds of one kind prepared, these compounds have strong affine with effective anticoagulin xa activity and to AT
Power.
WO9925720 discloses a kind of five sugar compounds with 4~6 sulfate radicals, and the reduction of sulfate groups is not
Antithrombotic activity is reduced, the decrease of platelet that heparin will not be caused to induce.
WO0224754 discloses five sugar compounds of one kind with biotin or biotin derivative covalent linkage.Biotin
Or derivatives thereof introducing there is no change pentasaccharides pharmaceutical activity, it is advantageous that such compound also has in emergency situations
The advantages of inhibiting its anticoagulant active, is neutralized by avidin solution agent rapidly.
WO2012172104 discloses five sugar compounds of high activity of a kind of half-life short, by the substitution for changing D unit
Base regulates and controls alkylation/O- sulphation pentasaccharides half-life period.
The half-life period of anticoagulation medicine is very important pharmacokinetic parameter.In clinic in the case where bleeding occurs
Need to close blood coagulation resisting function as quickly as possible, and the half-life period too long bleeding risk that will lead to increases.And for certain thrombus bolts
Plug property disease, such as pulmonary embolism, venae profunda conducted, non-ST elevation acute myocardial infraction, suitably extend the half-life period of anticoagulant
It is more favorable.
On the other hand, the core of anticoagulation medicine research and development is safety issue, how effectively to avoid hemorrhagic tendency, reduces out
Blood risk is the exploitation institute facing challenges of anticoagulation medicine.
Summary of the invention
In order to solve the problems in the existing technology, the present invention provides it is a kind of complete it is newly synthesized as shown in Formula II from
Five sugar compounds of sub- form or its acid or its salt:
Wherein, R1Selected from H, SO3 -Or C1~C4 alkyl；
R2Selected from H or SO3 -。
Preferably, five sugar compounds and its salt, anionic form are selected from such as flowering structure:
Wherein R1For C1~C4 alkyl, preferably methyl.
It is highly preferred that five sugar compounds and its salt, anionic form is selected from such as flowering structure:
Wherein R1For C1~C4 alkyl, preferably methyl.
The present invention includes synthesizing the sour form and its salt form of the pentasaccharides ,-COO in sour form-With-OSO3 -Functional group's difference
It is-COOH form and-OSO3H-shaped formula；Salt form is selected from sodium salt, sylvite, calcium salt or ammonium salt, particular certain cancers or sylvite.
Further, the present invention provides the preparation methods of five sugar compounds, prepare the method packet of II-1~II-3
Include following steps:
(1) disaccharides shown in formula DE is reacted into preparation formula DEFGH0 with trisaccharide shown in formula FGH5,
Wherein, RaFor Ac, Bn, C1~C4 alkyl；
(2) formula DEFGH0 is removed into benzyl with preparation formula DEFGH1,
Wherein, RbFor Ac, H, C1~C4 alkyl；
(3) sulfating reaction preparation formula DEFGH2 is occurred into for formula DEFGH1 depicted compound,
Wherein, RcFor Ac, SO3 -, C1~C4 alkyl；
(4) by compound hydrolysis preparation formula II-1~II-3 shown in formula DEFGH2.
Prepare the method for II-4~II-6 the following steps are included:
(1) trisaccharide shown in disaccharides shown in formula DE and formula FGH4 is reacted preparation formula DEFGH0-1,
Wherein, RaFor Ac, Bn, C1~C4 alkyl；
(2) formula DEFGH0-1 is removed into benzyl with preparation formula DEFGH1-1,
Wherein, RbFor Ac, H, C1~C4 alkyl；
(3) sulfating reaction preparation formula DEFGH2-1 is occurred into for formula DEFGH1-1,
Wherein, RcFor Ac, SO3 -, C1~C4 alkyl；
(4) formula DEFGH2-1 hydrolysis is prepared into pentasaccharides II-4~II-6.
The present invention also provides preparation five sugar compounds and its salt intermediate DE,
Wherein, RaFor Ac, Bn, C1~C4 alkyl, preferably RaFor Ac, Bn, methyl.
And following formula intermediate,
And following formula intermediate,
Wherein, RaFor Ac, Bn, C1~C4 alkyl；It is preferred that Ac, Bn, methyl.
And following formula intermediate,
Wherein, RbFor Ac, H, C1~C4 alkyl.
The present invention also provides following formula intermediate,
Wherein, RcFor Ac, SO3 -, C1~C4 alkyl.
On the other hand, the invention further relates to pharmaceutical composition, including containing the compounds of this invention as active constituent with
And optional pharmaceutically acceptable auxiliary material.
In each unit dose, five sugar compounds shown in Formula II of the invention, its acid or its salt are 0.1~100mg,
It is preferred that 0.5~50mg.
Described pharmaceutical composition can be administered by oral or extra-parenteral approach, and parenteral route includes but unlimited
In intravenous injection, intramuscular injection and subcutaneous injection.
For oral administration, pharmaceutical composition of the invention provides usually in the form of tablet, capsule, solution.
Tablet may include five sugar compounds shown in Formula II of the invention, its acid or its salt and pharmaceutically acceptable excipient.
The excipient includes but is not limited to diluent, disintegrating agent, adhesive, lubricant, sweetener, corrigent, colorant or anti-corrosion
Agent.Diluent can be selected from starch, lactose；Disintegrating agent can be selected from cornstarch, alginic acid；Adhesive can be selected from starch, povidone,
Gelatin；Lubricant can be selected from magnesium stearate, talcum powder, sodium stearyl fumarate.Capsule includes hard capsule and soft capsule.
In hard capsule, five sugar compounds shown in the Formula II of the invention as active constituent, its acid or its salt and diluent are mixed
It closes, in soft capsule, five sugar compounds shown in Formula II of the invention, its acid or its salt and water or oil such as peanut oil or olive
Oil mixing.
For parenteral route, pharmaceutical composition of the invention can by intravenous injection, intramuscular injection or
Subcutaneous administrations.It is usually provided with aseptic aqueous solution or suspension or freeze-dried powder, and adjusts suitable pH and isotonicity.
In addition, can optional auxiliary material such as mannitol, cellulose derivative, lecithin.Preferred administration route is subcutaneous administrations.
On the other hand, the invention further relates to five sugar compounds shown in Formula II, its acid or its salt preparation for prevent and/
Or the purposes in the drug of relevant to the disturbances of blood coagulation disease for the treatment of or morbid state.
In another aspect, the invention further relates to for preventing and/or treating disease relevant to blood coagulation disorder and disease
The method of state comprising given to individual in need five sugar compounds shown in Formula II of the invention, its acid or its salt or
Pharmaceutical composition of the invention.
Also on the one hand, the invention further relates to for preventing and/or treating disease relevant to blood coagulation disorder and disease
Five sugar compounds shown in the Formula II of the invention of state, its acid or its salt or pharmaceutical composition of the invention.
The disease relevant to coagulation function obstacle or the example of morbid state include but is not limited to venous thronbosis,
Especially Deep vain thrombosis, Arterial thrombosis, thrombophlebitis, pulmonary embolism, acute coronary syndrome, cardiac muscle
Infraction or apoplexy, and the artery occlusion as caused by thrombosis or embolism, angioplasty or thrombolysis process in or it
Artery afterwards blocks again, Postoperative Intravenous thrombosis or embolism, acute or chronic artery sclerosis, ST sections, Non-ST Elevation Acute cardiac muscle
Infarct.In haemodialysis and surgical operation, the compound of the present invention can also be used in the anticoagulant in extracorporeal circulation of blood.Also
The compound of the present invention can be used for the prevention of the relevant ischemic damage and reperfusion damage of solid organ transplantation.
Advantages of the present invention is mainly reflected in: the present invention has synthesized a series of novel by change E ring, the substituent group of G ring
Five sugar compounds, it is amazing be five sugar compounds of the invention have unexpected technical effect.Firstly, of the invention
Five sugar compounds have compared with the stronger anticoagulin xa activity of Fondaparinux sodium, dosage is smaller and less side effects；
Secondly, five sugar compounds of the invention are extended compared with the Half-life in vivo of Fondaparinux sodium, it can achieve every 2~3 days and be administered
Once, it is especially suitable for the treatment of pulmonary embolism, venae profunda conducted, non-ST elevation acute myocardial infraction patient；It is commented in preliminary safety
In estimating, the compounds of this invention does not almost make significant difference to hemorrhagic tendency, shows important potential using value；Furthermore this hair
The preparation method of bright compound greatly simplifies, and manufacturing cost is low, significantly reduces the development cost of bulk pharmaceutical chemicals, is suitable for extensive
Industrialized production.
Specific embodiment
Present inventor has synthesized five saccharide compounds shown in general formula II, and given birth to by further investigation
Object experimental study finds that five saccharide compounds of the invention have high anticoagulin xa activity and suitably disappear in vivo
Except half-life period, be especially suitable for anticoagulant.
Definition
Unless stated to the contrary, following that there are following meanings with term in the specification and in the claims
" alkyl " refers to the aliphatic hydrocarbon group of saturation, the saturation monovalent hydrocarbon including linear chain or branched chain, and the alkyl has institute
The carbon atom number shown.If term " C1~C4 alkyl " includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, suitable alkyl base
Group includes methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, tert-butyl.
Claims and abbreviation used in the description its meaning see the table below:
| Bz | Benzoyl |
| Bn | Benzyl |
| Ac | Acetyl group |
| DMAP | 4-dimethylaminopyridine |
| DBU | 11 carbon -7- alkene of 1,8- diazabicylo |
| TEMPO | 2,2,6,6- tetramethyl piperidine-nitrogen-oxide |
| BAIB | Iodobenzene diethylester |
| TCCA | Sym-closene |
| TMSOTf | Trimethylsilyl trifluoromethanesulfonate |
| Dowex | Ion exchange resin |
| Sephadex | Sephadex |
Preparation method:
The preparation method of five sugar compounds of the invention is described more particularly below, but these specific preparation methods are not right
The scope of the present invention constitutes any restrictions.In addition, reaction condition such as reactant, solvent, alkali, the amount of compound used therefor, reaction temperature
Degree, reaction time etc. are not limited to following example.
Five sugar compounds of the invention can also optionally will be describing or known in the art various in the present specification
Synthetic method combines and is conveniently made, and such combination can be easy to carry out by those skilled in the art.
The synthesis of first part's monosaccharide D ring
Synthetic route:
A) dimethyl suflfate, KOH, acetonitrile, b) acetic acid, sulfuric acid, water, c) Tritox, DBU, methylene chloride
The preparation of compound D1: D0 (748g) is dissolved in tetrahydrofuran, excessive potassium hydroxide does alkali, sulfuric acid is added dropwise at 0 DEG C
Dimethyl ester reacts 2h, has reacted plus 4h is quenched in potassium hydroxide aqueous solution, and then ethyl acetate extracts liquid separation, is spin-dried for obtaining D1
(804g), yield 100%.
The preparation of compound D2: acetic acid: water is added in D1 (804g): in sulfuric acid=2000g:400g:98g solution, returned
Add ethyl acetate and water extraction after stream reaction 1h, organic phase is evaporated to obtain D2 (690g), yield 89%.
The preparation of compound D3: being dissolved in anhydrous methylene chloride for D2 (388g), and Tritox 288g, DBU 15g is added to react
Then 1h revolves dry chromatography and obtains D3 (490g), yield 92%.
The preparation of second part monosaccharide E ring
Synthetic route:
A) dimethyl suflfate, KOH, tetrahydrofuran, b) trifluoracetic acid, water, c) para-methylbenzenepyridinsulfonate sulfonate, benzaldehyde diformazan
Acetal, d) ethyl acetate, aceticanhydride, triethylamine, DMAP, e) boron trifluoride ether, p methoxy phenol, f) potassium hydroxide, g) system
Standby E7: potassium hydroxide, benzyl bromine prepare E8: potassium hydroxide, dimethyl suflfate, h) acetic acid, water, i) TEMPO, BAIB, dichloromethane
Alkane, water, j) iodomethane, saleratus, acetonitrile
The preparation of compound E1~E4: Tetrahedron, 2012,68 (36), 7386-7399. are referred to
The preparation of compound E5: E4 (366g) and p methoxy phenol (130g) are dissolved in 366ml anhydrous methylene chloride,
Boron trifluoride ether 280g is injected at room temperature, and add methylene chloride dilution after reaction 2h, is quenched with saturated sodium bicarbonate aqueous solution, so
Liquid separation afterwards, organic phase is dry with anhydrous sodium sulfate, obtains E5 (387g), yield 90%.
The preparation of compound E6: being dissolved in 860ml methanol for intermediate E 5 (86g), sodium methoxide 2.7g be added, and is stirred at room temperature anti-
Acetic acid on the rocks is answered after 1h to be neutralized to neutrality, evaporated under reduced pressure, residue adds methylene chloride 1L, water 1L washing, and liquid separation is spin-dried for organic phase
It obtains E6 (77.6g), yield 100%.
The preparation of compound E7: E6 (38.8g) is dissolved in 400ml acetonitrile, adds potassium hydroxide 23g, sulfuric acid two is added dropwise at 10 DEG C
It depressurizes and is spin-dried for after methyl esters 18.9g, reaction 4h, residue adds ethyl acetate 1L, water 1L extracting and washing, and organic phase decompression is spin-dried for slightly
Product 48g, crude product ethyl acetate, petroleum ether recrystallize to obtain E7 (36.4g), yield 91%.
The preparation of compound E8: E5 (98.5g) is dissolved in 430ml acetic acid, is added water (86g), 70 DEG C be stirred to react 1h after subtract
Pressure revolving is dry, adds water, ethyl acetate, is adjusted to pH=7 with sodium carbonate, then liquid separation obtains organic phase, and organic phase evaporated under reduced pressure obtains
E8 crude product 80g, column chromatograph to obtain E8 sterling 70.5g, yield 90%.
The preparation of compound E9: E7 (36g) is dissolved in 450ml acetic acid, and water 90g is added, and 70 DEG C are stirred to react decompression rotation after 1h
It is evaporated, adds water, ethyl acetate, be adjusted to pH=7 with sodium carbonate, then liquid separation, organic phase evaporated under reduced pressure obtains E9 crude product 38g, column layer
Analyse to obtain sterling 26.7g, yield 95%.
The preparation of compound E10: E8 (68.4g) is added in methylene chloride (342ml) and water (342ml), is added
TEMPO0.63g and iodobenzene diethylester 86g is vigorously stirred room temperature reaction 45min, and then plus 30% sodium sulfite aqueous solution is quenched.
Water phase is extracted with ethyl acetate, and organic phase is dry with anhydrous sodium sulfate, and evaporated under reduced pressure obtains E10 (57.0g), yield 80%.
The preparation of compound E11: it with reference to the preparation of E10, with E9 (33g) for raw material, is made E11 (28g, yield 81%).
The preparation of compound E12: E10 (57.6g) is dissolved in acetonitrile 576ml, and potassium carbonate 33.5g and iodomethane 34.5g is added, is stirred to react
8h, has reacted plus triethylamine is quenched, and saturated common salt water washing three times, makes pH=7~8, and decompression is spin-dried for organic phase.Column chromatographic purifying
It obtains E12 (51g), yield 85%.1H NMR(300MHz,CDCl3) δ 6.99 (m, 2H), 6.84 (m, 2H), 6.2 (d, J=3.6Hz,
1H), 5.47 (dd, J=9.2,3.6Hz, 1H), 4.74 (s, 1H), 4.53 (m, 1H), 4.43 (d, 1H), 3.99 (t, 1H), 3.81
(m,3H),3.70(s,3H),3.41(s,3H),2.02(s,3H).MS(ESI):393.2[M+Na]+
The preparation of compound E13: with reference to the preparation method of E12, with E11 (26g) for raw material, E13 (25g, yield is made
92%).1H NMR(300MHz,CDCl3) δ 6.99 (m, 2H), 6.84 (m, 2H), 5.80 (d, J=3.6Hz, 1H), 4.74 (s,
1H),4.53(d,1H),4.43(m,1H),4.10(m,1H),3.81(m,3H),3.70(s,3H),3.60(m,1H),3.41(s,
3H).MS(ESI):365.2[M+Na]+
The preparation of Part III monosaccharide F ring and H ring
Synthetic route:
A) sulfuric acid, benzaldehyde, DMF, b) benzyl bromine, potassium hydroxide, acetonitrile, c) triethylsilane, boron trifluoride ether, dichloro
Methane, d) acetic acid, sulfuric acid, water, e) aceticanhydride, triethylamine, DMAP, ethyl acetate, f) piperidines, tetrahydrofuran, g) Tritox,
DBU, methylene chloride
The preparation of compound F1 (H1)-F3 (H3): referring to Bioorganic&Medicinal Chemistry Letters,
2009,19(14),3875-3879。
The preparation of compound F4: being added acetic acid 2.3kg and water 464g for F3 (464g), and sulfuric acid 46g is added, and 100 DEG C anti-
Answer 1h.Evaporated under reduced pressure has been reacted, F4 (427.5g, yield 95%) is obtained.
The preparation of compound F5: ethyl acetate dissolution is added in F4 (400g), triethylamine 200ml is added, aceticanhydride is added dropwise
200ml adds saturated aqueous sodium carbonate to neutralize after reacting 1h, and ethyl acetate extraction, evaporated under reduced pressure obtains F5 (475g, yield
100%).1H NMR(300MHz,CDCl3) δ 7.35-7.29 (m, 15H), 6.0 (d, J=3.6Hz, 1H), 4.9 (dd, J=9.2,
3.4Hz,1H),4.7-4.6(m,6H),4.4(m,1H),4.27-4.18(m,2H),3.6(d,1H),3.4(d,1H),2.19(s,
3H),2.14(s,3H).MS(ESI):557.3[M+Na]+
The preparation of compound F6: tetrahydrofuran dissolution is added in F5 (450g), piperidinyl-1 43g is added, adds acetic acid after reacting 6h
It neutralizes, vacuum rotary steam, residue ethyl acetate is dissolved, washed, and evaporated under reduced pressure organic phase obtains 373g, yield 90%.
The preparation of compound F7: being added methylene chloride dissolution for F6 (350g), Tritox 400g and DBU 10g be added,
It is spin-dried for after reaction 1h, column chromatography obtains F7 (406g, yield 90%).
Part IV: the synthesis of monosaccharide G ring
Synthetic route:
A) dimethyl suflfate, KOH, tetrahydrofuran, b) phosphomolybdic acid, silica gel, water, acetonitrile, c) dipotassium hydrogen phosphate trihydrate, phosphorus
Acid dihydride potassium, water, TEMPO, NaClO2, KBr, TCCA, d) iodomethane, potassium carbonate, acetonitrile, e) trifluoromethanesulfanhydride anhydride, pyridine, f)
Sodium trifluoroacetate, DMSO, water, g) trifluoracetic acid, water, h) aceticanhydride, triethylamine, DMAP, ethyl acetate, j) trifluoromethanesulfonic acid neodymium,
K) Tritox, DBU, methylene chloride
The preparation of compound G1: referring to the synthesis of E1.
The preparation of compound G2: being dissolved in acetonitrile 9.72kg for G1 (1.5kg), add silica gel 0.36kg, phosphomolybdic acid 0.036kg,
Water 0.8kg, is stirred at room temperature 16h, filters off silica gel, is neutralized to pH=7, filtrate decompression revolving with sodium carbonate liquor, residue add water,
Ethyl acetate extraction, filtrate decompression are evaporated to obtain G2 (1.15kg, yield 90%).
The preparation of compound G3: being dissolved in tetrahydrofuran 10L for G2 (1.15kg), be added dipotassium hydrogen phosphate trihydrate 500g,
Potassium dihydrogen phosphate 500g, water 1.5kg, TEMPO 20g, NaClO2300g, KBr 50g, TCCA 500g, react 1h after plus it is anhydrous
Sodium sulfite is quenched, and then decompression is spin-dried for, and residue is washed with ethyl acetate, and organic phase decompression is spin-dried for obtaining G3 (1.1kg, yield
90%).
The preparation of compound G4: G3 (1.1kg) is dissolved in acetonitrile 6.0kg, 1.22kg potassium carbonate, iodomethane is added
Reaction 8h is stirred at room temperature in 0.73kg, after completion of the reaction, triethylamine is added, iodomethane is quenched and filters, filtrate decompression is spin-dried for obtaining G4
(929g, yield 80%).
The preparation of compound G5: pyridine 1L and acetonitrile 5L is added in G4 (885g), trifluoromethanesulfanhydride anhydride 950g is added dropwise, is added dropwise
Reaction 1h is finished, then elutriation crystalline substance on the rocks filters, and filter cake is dissolved with methylene chloride, and then methylene chloride is mutually washed with water, dries, and subtracts
Pressure is spin-dried for obtaining G5 (1280g, yield 96%).
The preparation of compound G6: G5 (1280g) and sodium trifluoroacetate 1kg are dissolved in DMSO 10L and water 1L, stir 8h
Afterwards plus ethyl acetate extraction, decompression are spin-dried for obtaining G6 (808g, yield 95%).
The preparation of compound G7: G6 (808g) is added in trifluoracetic acid 2L and water 200ml, is depressurized and is spin-dried for after stirring 1h
G7 (547g, yield 80%).
The preparation of compound G8: being added methylene chloride 5L, DMAP 50g and triethylamine 1.15kg for G7 (547g), and then 0
Aceticanhydride 1.15kg is added dropwise at DEG C, adds water washing after reacting 0.5h, methylene chloride, which mutually depressurizes, is spin-dried for obtaining G8 (815g, yield 95%).
The preparation of compound G9: by G8 (810g) be added methanol 12L, be added trifluoromethanesulfonic acid neodymium 60g, stir 12h after it is dense
Contracting, column chromatography, obtains G9 (605g, yield 85%).
The preparation of compound G10: being added methylene chloride 6kg for G9 (605g), Tritox 1.2kg and DBU 45g be added,
It is spin-dried for after reaction 1h, column chromatography obtains G10 (801g, yield 90%).
The synthesis of Part V disaccharides DE
Synthetic route 1:
A) TMSOTf, methylene chloride, b) palladium carbon, hydrogen, c) trityl group chlorine, triethylamine, DMAP, d) chlorobenzoyl chloride,
Triethylamine, e) acetic acid, water, f) benzyl bromine, potassium hydroxide, g) ammonium ceric nitrate, h) Tritox, DBU, methylene chloride
The preparation of compound DE1: it takes D3 (43g) and E12 (25g) to be dissolved in anhydrous methylene chloride, is added dropwise under nitrogen protection
After TMSOTf 1g, -20 DEG C of reaction 1h plus triethylamine is quenched, and decompression is spin-dried for, and column chromatography obtains DE1 αisomer 38g, yield 76%.
The preparation of compound DE2: it takes D3 (56g) and E13 (30g) to be dissolved in anhydrous methylene chloride, is added dropwise under nitrogen protection
After TMSOTf 1g, -20 DEG C of reaction 1h plus triethylamine is quenched, and decompression is spin-dried for, and column chromatography obtains DE2 αisomer 46g, yield 74%.
The preparation of compound DE3: being dissolved in anhydrous methanol for DE1 (38g), and 10% palladium carbon, reductase 12 4h under hydrogen normal pressure is added
Afterwards, it filters, is spin-dried for obtaining DE3 (28.8g), yield 100%.
The preparation of compound DE4: being dissolved in anhydrous methanol for DE2 (46g), and 10% palladium carbon, reductase 12 4h under hydrogen normal pressure is added
Afterwards, it filters, is spin-dried for obtaining DE4 (34.3g), yield 100%.
The preparation of compound DE5: DE3 (28.8g) is added methylene chloride dissolution, and trityl chloride 15g, DMAP 5g is added
With triethylamine 30ml, it is stirred to react 8h, is spin-dried for, column chromatography obtains 41g, yield 90%.
The preparation of compound DE6: DE4 (34.3g) is added methylene chloride dissolution, and trityl chloride 19g, DMAP 5g is added
With triethylamine 30ml, it is stirred to react 8h, is spin-dried for, column chromatography obtains 46g, yield 92%.
The preparation of compound DE7: DE5 (41g) is dissolved in pyridine, -20 DEG C of coolings add chlorobenzoyl chloride 7.2g, after reacting 4h
Add saturated sodium bicarbonate aqueous solution crystallization, filter, filter cake dries to obtain 46.3g, yield 100%.
The preparation of compound DE8: DE6 (46g) is dissolved in pyridine, -20 DEG C of coolings add chlorobenzoyl chloride 8.4g, after reacting 4h
Add saturated sodium bicarbonate aqueous solution crystallization, filter, filter cake dries to obtain 52.2g, yield 100%.
The preparation of compound DE9: DE7 (46g) is dissolved in the mixed solution of 230g acetic acid and 46g water, 70 DEG C of reaction 1h, rotation
Dry, column chromatography obtains 33.7g, yield 98%.
DE8 (52g): being dissolved in the mixed solution of 260g acetic acid and 52g water by the preparation of compound DE10,70 DEG C of reaction 1h,
It is spin-dried for, column chromatography obtains 37.7g, yield 85%.
The preparation of compound DE11: being dissolved in acetonitrile 330ml for DE9 (33g), 35g potassium carbonate be added, and is added dropwise bromobenzyl 20g, and 0
DEG C reaction 2h, adds water and ethyl acetate to extract, is spin-dried for organic phase, column chromatography obtains 36.7g, yield 98%.1H NMR(300MHz,
CDCl3) δ 8.04-7.55 (m, 5H), 7.34-7.29 (m, 5H), 6.99-6.84 (m, 4H), 6.39 (d, J=3.6Hz, 1H),
5.99 (d, J=3.6Hz, 1H), 5.47 (dd, 1H), 5.17 (dd, J=9.2,3.4Hz, 1H), 4.65-4.61 (m, 3H),
4.23-4.19(m,3H),4.00(m,1H),3.81(s,3H),3.70(s,3H),3.61(d,1H),3.50(d,1H),3.41
(m,9H),3.36(d,1H),2.02(s,3H).MS(ESI):777.3[M+Na]+
The preparation of compound DE12: being dissolved in acetonitrile 370ml for DE10 (37g), 50g potassium carbonate be added, and is added dropwise bromobenzyl 25g, and 0
DEG C reaction 2h, adds water and ethyl acetate to extract, is spin-dried for organic phase, column chromatographs to obtain 41.8g, yield 99%.1H NMR(300MHz,
CDCl3) δ 8.04-7.55 (m, 5H), 7.34-7.29 (m, 5H), 6.99-6.84 (m, 4H), 5.99 (d, J=3.6Hz, 1H),
5.97 (d, J=3.6Hz, 1H), 5.17 (dd, J=9.2,3.4Hz, 1H), 4.65-4.61 (m, 3H), 4.23-4.19 (m, 2H),
4.10(dd,1H),4.00(m,1H),3.81-3.80(m,4H),3.70(s,3H),3.61(d,1H),3.50(d,1H),3.41
(m,12H),3.36(d,1H).MS(ESI):749.3[M+Na]+
DE11 (36g): being dissolved in the mixed solution of acetonitrile 0.4L, toluene 0.2L and water 0.2L by the preparation of compound DE13,
Ammonium ceric nitrate 50g is added, after reacting at room temperature 1h, ethyl acetate extraction, decompression is spin-dried for, and obtains DE13 (29.1g), yield 94%.Change
It closes the preparation of object DE14: DE12 (41g) is dissolved in the mixed solution of acetonitrile 0.4L, toluene 0.2L and water 0.2L, cerous nitrate is added
Ammonium 50g, after reacting at room temperature 1h, ethyl acetate extraction, decompression is spin-dried for, and obtains DE14 (31.5g), yield 90%.Compound DE15's
Preparation: being dissolved in anhydrous methylene chloride for DE13 (29g), Tritox 30g and DBU2.9g be added, after reacting at room temperature 1h, decompression rotation
Dry doubling column chromatography, obtains DE15 (31.9g), yield 90%.
The preparation of compound DE16: being dissolved in anhydrous methylene chloride for DE14 (31g), be added Tritox 31g and
DBU3.1g, after reacting at room temperature 1h, decompression is spin-dried for and column chromatography, obtains DE16 (34.4g), yield 90%.
Synthetic route 2:
A) potassium carbonate, methanol, b) tribromo-acetyl imines benzyl ester, trifluoromethanesulfonic acid, c) ammonium ceric nitrate, d) Tritox,
DBU, methylene chloride
The preparation of compound DE17: being dissolved in methanol 1L for DE11 (75.4g), potassium carbonate 150g be added, after being stirred to react 8h,
It is spin-dried for, adds ethyl acetate, water extraction, be spin-dried for organic phase and obtain DE17 (64g), yield 90%.
The preparation of compound DE18: DE17 (64g) is dissolved in anhydrous methylene chloride 1L, tribromo-acetyl imines acid benzyl ester is added
25g and trifluoromethanesulfonic acid 1g is added triethylamine neutralization after reacting 1h, is spin-dried for, crude product column chromatographs to obtain DE18 (65g), yield 90%
。1H NMR(300MHz,CDCl3)δ8.04-7.55(m,5H),7.34-7.29(m,10H),6.99-6.84(m,4H),,6.00
(d, J=3.6Hz, 1H), 5.99 (d, J=3.6Hz, 1H), 5.17 (dd, J=9.2,3.4Hz, 1H), 4.65-4.61 (m, 5H),
4.23 (s, 1H), 4.19 (d, 1H), 4.10 (dd, 1H), 4.00 (m, 1H), 3.81-3.80 (m, 4H), 3.70 (s, 3H), 3.61
(d,2H),3.50(d,1H),3.41(m,,9H).MS(ESI):825.3[M+Na]+
The preparation of compound DE19: DE18 (60g) is dissolved in the mixed solution of acetonitrile 0.6L, toluene 0.4L and water 0.4L, adds
Enter ammonium ceric nitrate 100g, after reacting at room temperature 1h, ethyl acetate extraction, decompression is spin-dried for obtaining DE19 (42g), yield 80%.
The preparation of compound DE20: DE19 (42g) is dissolved in anhydrous methylene chloride 500ml, and Tritox 50g and DBU is added
4g is spin-dried for after reacting 1h, and column chromatography obtains DE20 (41g), yield 81%.
The synthesis of Part VI trisaccharide FGH
Synthetic route:
A) TMSOTf, methylene chloride, b) sodium methoxide, methanol, c) TMSOTf, methylene chloride, d) potassium carbonate, carbinol compound
The preparation of GH0: G10 (631g) and H3 (465g) are dissolved in anhydrous methylene chloride, TMSOTf 23g is added dropwise at 0 DEG C, after reacting 1h
Triethylamine is added to be quenched, column chromatographs to obtain GH0 (677g), yield 90%.
The preparation of compound GH1/GH2: being dissolved in methanol 4L for GH0 (377g), potassium carbonate 70g be added, be spin-dried for after reacting 2h,
Add water, ethyl acetate extraction, washing is evaporated, column chromatographs to obtain GH1 (154g, yield 46%) and GH2 (142g, yield 40%).
The preparation of compound FGH1: GH1 (66.8g) and H3 (93.6g) are dissolved in anhydrous methylene chloride, are added dropwise at 0 DEG C
TMSOTf 2.2g adds triethylamine to be quenched after reacting 1h, and column chromatographs to obtain FGH1 (91.4g, yield 80%).
The preparation of compound FGH2: GH2 (71.1g) and H3 (93.6g) are dissolved in anhydrous methylene chloride, are added dropwise at 0 DEG C
TMSOTf 2.2g adds triethylamine to be quenched after reacting 1h, and column chromatographs to obtain FGH2 (93.6g, yield 79%).
The preparation of compound FGH3: being dissolved in anhydrous methylene chloride for FGH1 (90g) and tribromo-acetyl imines acid benzyl ester, and 0 DEG C
Lower dropwise addition trifluoromethanesulfonic acid 10g adds triethylamine to be quenched after reacting 1h, and column chromatographs to obtain FGH3 (77.7g, yield 80%).
The preparation of compound FGH4: being dissolved in methanol 1000ml for FGH2 (50g) and potassium carbonate 20g, adds 80% after stirring 2h
Aqueous acetic acid is quenched, and is spin-dried for, and column chromatography obtains 33.8g, yield 70%.
1H NMR(300MHz,CDCl3) δ 7.34-7.29 (m, 30H), 5.99 (d, J=3.6Hz, 1H), 5.60 (m, 2H),
5.17 (dd, J=9.0,3.4Hz, 1H), 4.88 (s, 1H), 4.64-4.62 (m, 13H), 4.23-4.19 (m, 2H), 4.00 (m,
1H),3.80-3.77(m,3H),3.70(s,3H),3.61(d,2H),3.50(d,1H),3.40(d,6H),3.36(s,1H),
2.02(s,3H).MS(ESI):1165.5[M+Na]+
The preparation of compound FGH5: being dissolved in methanol 1000ml for FGH4 (50g) and potassium carbonate 20g, adds 80% after stirring 2h
Aqueous acetic acid is quenched, and is spin-dried for, and column chromatographs to obtain 38.6g, yield 80%.
1H NMR(300MHz,CDCl3)δ7.34-7.29(m,30H),5.60(m,3H),4.88(s,1H),4.64–4.62
(m,13H),4.23(m,1H),4.00(m,1H),3.80-3.77(m,7H),3.70(s,3H),3.61-3.60(m,3H),3.50
(d,1H),3.41-3.36(m,8H).MS(ESI):1213.5[M+Na]+
1 pentasaccharides II-1 (R of embodiment1For methyl, sodium salt) preparation
Synthetic route:
The preparation of compound DEFGH10: DE16 (32g) and FGH5 (50g) plus anhydrous methylene chloride are dissolved, and are dripped at 0 DEG C
Add TMSOTf 1g, add triethylamine to be quenched after reacting 1h, column chromatography obtains DEFGH10 (61g, yield 81%).1H NMR
(300MHz,CDCl3)δ8.05-7.55(m,5H),7.34-7.29(m,40H),5.99(d,1H),5.60(m,4H),5.17
(dd, J=9.0,3.4Hz, 1H), 4.64-4.62 (m, 18H), 4.23-4.19 (m, 3H), 4.00 (m, 3H), 3.80-3.77 (m,
8H),3.70(s,6H),3.61(d,3H),3.50(d,3H),3.41-3.40(m,18H),3.36(m,3H).MS(ESI):
1815.8[M+Na]+
The preparation of compound DEFGH11: DEFGH10 (60g) is dissolved in anhydrous methanol, 10% palladium carbon, hydrogen normal pressure is added
Filtering is spin-dried for obtaining DEFGH11 (34g, yield 95%) after reductase 12 4h.1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),
5.99 (d, J=3.6Hz, 1H), 5.60 (d, J=8Hz, 1H), 5.40 (m, 3H), 5.17 (dd, J=9.0,3.4Hz, 1H),
4.77(d,3H),4.71(d,2H),4.64–4.62(m,2H),4.23-4.19(m,3H),4.10(m,1H),3.94-3.80(m,
12H),3.70(d,6H),3.57-3.50(m,8H),3.41-3.40(m,18H),3.30(m,2H).MS(ESI):1095.4[M+
Na]+
The preparation of compound DEFGH12: n,N-Dimethylformamide dissolution is added in DEFGH11 (5g), is added excessive three
Sulfur oxide triethylamine salt, 50 DEG C of reactions add saturated sodium bicarbonate aqueous solution to be quenched, are concentrated under reduced pressure afterwards for 24 hours.Gel column (Sephadex
G-25 it) separates, eluent (water/acetonitrile=4/1, v/v).Sodium form ion exchange resin column (DOWEX-50Na), pure water elution, subtracts
Pressure is concentrated to get colorless solid DEFGH12 (7.8g, yield 89%).1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),
5.99 (d, J=3.6Hz, 1H), 5.60 (d, J=8Hz, 1H), 5.54 (m, 3H), 5.25-5.24 (m, 6H), 4.64-4.62
(m,2H),4.23-4.19(m,6H),3.96-3.94(m,6H),3.80(m,2H),3.74(m,1H),3.70(d,6H),3.50-
3.44(m,3H),3.41-3.40(m,18H).MS(ESI):921.0[(M-2Na)/2]-
The preparation of pentasaccharides II-1 (sodium salt): 4N NaOH aqueous solution is added in DEFGH12 (6.3g), room temperature reaction subtracts afterwards for 24 hours
Pressure concentration.Gel column (Sephadex G-25) separation, eluent (water/acetonitrile=4/1, v/v).Sodium form ion exchange resin column
(DOWEX-50Na), pure water elutes, and is concentrated under reduced pressure to give colorless solid (5.2g, yield 87%).1H NMR(300MHz,CDCl3)
δ5.60(d,1H),5.54(m,3H),5.40(m,1H),5.30(m,1H),5.25-5.24(m,4H),4.77(m,1H),4.64–
4.62(m,2H),4.23-4.21(m,5H),4.10(m,2H),3.96-3.94(m,6H),3.74(m,1H),3.60(m,2H),
3.50-3.40(m,18H).MS(ESI):876.9[(M-2Na)/2]-
The preparation of 2 pentasaccharides II-2 (sodium salt) of embodiment
Synthetic route:
Referring to the preparation method of embodiment 1, react using DE15 and FGH5 as raw material, then remove benzyl through hydrogenolysis
Pentasaccharides II-2 (sodium salt) is made in base, sulfating reaction, hydrolysis.
Compound DEFGH20:1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),7.34-7.29(m,40H),
5.99 (d, 1H), 5.97 (d, J=3.4Hz, 1H), 5.60 (m, 3H), 5.17 (dd, J=9.0,3.4Hz, 2H), 4.64-4.62
(m,18H),4.23-4.19(m,4H),4.00(m,3H),3.80-3.77(m,6H),3.70(s,6H),3.61(d,3H),3.50
(d,3H),3.41-3.40(m,18H),3.36(m,2H),2.02(s,3H).MS(ESI):1843.8[M+Na]+
Compound DEFGH21:1H NMR(300MHz,CDCl3) δ 8.05-7.55 (m, 5H), 5.99 (d, J=3.6Hz,
2H), 5.40 (m, 3H), 5.17 (dd, J=9.0,3.4Hz, 2H), 4.77 (d, 3H), 4.71 (d, 2H), 4.64-4.62 (m,
2H),4.23-4.19(m,4H),4.10(m,1H),3.94-3.80(m,10H),3.70(d,6H),3.57-3.50(m,8H),
3.41-3.40(m,15H),3.30(m,2H),2.02(s,3H).MS(ESI):1123.4[M+Na]+
Compound DEFGH22:1H NMR(300MHz,CDCl3)δ5.54(m,3H),5.40(m,2H),5.25-5.24(m,
5H),4.77(m,2H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,2H),3.96-3.94(m,6H),
3.74(m,1H),3.70(m,6H),3.60(m,2H),3.50-3.40(m,18H).MS(ESI):861.9[(M-2Na)/2]-
Pentasaccharides II-2 (sodium salt):1H NMR(300MHz,CDCl3)δ5.54(m,3H),5.40(m,2H),5.25-5.24(m,
5H),4.77(m,2H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,2H),3.96-3.94(m,6H),
3.75(m,1H),3.60(m,2H),3.50-3.40(m,18H).MS(ESI):869.9[(M-2Na)/2]-
The preparation of 3 pentasaccharides II-3 (sodium salt) of embodiment
Synthetic route:
It referring to the method for embodiment 1, reacts using DE20 and FGH5 as raw material, then removes benzyl, sulphur through hydrogenolysis
Pentasaccharides II-3 (sodium salt) is made in acidification reaction, hydrolysis.
Compound DEFGH30:1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),7.34-7.29(m,45H),
5.99 (d, 1H), 5.60 (m, 4H), 5.17 (dd, J=9.0,3.4Hz, 1H), 4.64-4.62 (m, 20H), 4.23-4.19 (m,
3H),4.00(m,3H),3.80-3.77(m,8H),3.70(s,6H),3.61(d,3H),3.50(d,3H),3.41-3.40(m,
15H),3.36(m,3H).MS(ESI):1893.8[M+Na]+
Compound DEFGH31:1H NMR(300MHz,CDCl3) δ 8.05-7.55 (m, 5H), 5.99 (d, J=3.6Hz,
2H), 5.40 (m, 4H), 5.17 (dd, J=9.0,3.4Hz, 1H), 4.77 (d, 4H), 4.71 (d, 2H), 4.64-4.62 (m,
2H),4.23-4.19(m,3H),4.10(m,2H),3.94-3.80(m,10H),3.70(d,6H),3.60-3.57(m,5H),
3.51-3.50(m,4H),3.41-3.40(m,15H),3.30(m,2H).MS(ESI):1081.4[M+Na]+
Compound DEFGH32:1H NMR(300MHz,CDCl3) δ 8.05-7.55 (m, 5H), 5.99 (d, J=3.6Hz,
1H),5.54(m,4H),5.30(m,2H),5.25-5.17(m,5H),4.64–4.62(m,2H),4.23-4.19(m,6H),
3.96-3.94(m,6H),3.74(m,2H),3.70(m,6H),3.60(m,2H),3.50-3.40(m,18H).MS(ESI):
964.9[(M-2Na)/2]-
Pentasaccharides II-3 (sodium salt):1H NMR(300MHz,CDCl3)δ5.54(m,4H),5.40(m,1H),5.30-5.24(m,
6H),4.77(m,1H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,1H),3.96-3.94(m,6H),
3.74(m,2H),3.60(m,1H),3.50-3.40(m,18H).MS(ESI):920.9[(M-2Na)/2]-
The preparation of 4 pentasaccharides II-4 (sodium salt) of embodiment
Synthetic route:
It referring to the method for embodiment 1, is reacted by raw material of DE15 and FGH4, then removes benzyl, sulphation through hydrogenolysis
Pentasaccharides II-4 (sodium salt) is made in reaction, hydrolysis.
Compound DEFGH40-1:1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),7.34-7.29(m,35H),
5.99(d,3H),5.60(m,2H),5.17(m,3H),4.64–4.62(m,16H),4.23-4.19(m,5H),4.00(m,3H),
3.80-3.77(m,4H),3.70(s,6H),3.61(d,3H),3.50(d,3H),3.41-3.40(m,15H),3.36(m,3H)
.MS(ESI):1795.7[M+Na]+
Compound DEFGH41-1:1H NMR(300MHz,CDCl3) δ 8.05-7.55 (m, 5H), 5.99 (d, J=3.6Hz,
3H),5.40(m,2H),5.17(m,3.4Hz,3H),4.77(m,3H),4.71(d,1H),4.64–4.62(m,2H),4.23-
4.19(m,5H),3.94-3.80(m,10H),3.70(d,6H),3.57-3.50(m,7H),3.41-3.40(m,15H),3.30
(m,2H),2.02(d,6H).MS(ESI):1175.4[M+Na]+
Compound DEFGH42-1:1H NMR(300MHz,CDCl3)δ8.07-7.55(m,5H),5.54(m,3H),5.36
(m,4H),5.09(m,2H),4.75–4.71(m,5H),4.21(m,3H),4.06-3.86(m,11H),3.68(m,6H),3.50
(s,3H),3.30-3.26(m,13H),3.308(m,2H),2.21(d,6H).MS(ESI):905.0[(M-2Na)/2]-
Pentasaccharides II-4:1H NMR(300MHz,CDCl3)δ5.54(m,2H),5.40(m,3H),5.30-5.24(m,4H),
4.77(m,3H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,3H),3.96-3.94(m,6H),3.60(m,
3H),3.50-3.40(m,18H).MS(ESI):818.9[(M-2Na)/2]-
The preparation of 5 pentasaccharides II-5 of embodiment (R1 is methyl, sodium salt)
Synthetic route:
It referring to the method for embodiment 1, is reacted by raw material of DE16 and FGH4, then removes benzyl, sulphation through hydrogenolysis
Pentasaccharides II-5 (sodium salt) is made in reaction, hydrolysis.
The preparation of compound DEFGH50-1:1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),7.34-7.29
(m,35H),5.99(d,2H),5.60(m,3H),5.17(m,2H),4.64–4.62(m,16H),4.23-4.19(m,4H),
4.00(m,3H),3.80-3.77(m,6H),3.70(s,6H),3.61(d,3H),3.50(d,3H),3.41-3.40(m,18H),
3.36(m,3H).MS(ESI):1767.7[M+Na]+
The preparation of compound DEFGH51-11H NMR(300MHz,CDCl3) δ 8.05-7.55 (m, 5H), 5.99 (d, J=
3.6Hz,3H),5.60(m,1H),5.40(m,2H),5.17(m,3.4Hz,2H),4.77(m,2H),4.71(d,2H),4.64–
4.62(m,2H),4.23-4.19(m,4H),3.94-3.80(m,12H),3.70(d,6H),3.57-3.50(m,7H),3.41-
3.40(m,18H),3.30(m,2H),2.02(d,3H).MS(ESI):1137.4[M+Na]+
The preparation of compound DEFGH52-1:1H NMR(300MHz,CDCl3) δ 8.07-7.55 (m, 5H), 5.99 (d, J=
3.6Hz,2H),5.60(m,1H),5.54(m,2H),5.24-5.17(m,6H),4.63(m,2H),4.23–4.19(m,7H),
3.96-3.94(m,6H),3.80(m,2H),3.70(m,6H),3.50-3.41(m,21H),2.02(s,3H).MS(ESI):
891.0[(M-2Na)/2]-
The preparation of pentasaccharides II-5:1H NMR(300MHz,CDCl3)δ5.60(m,1H),5.54(m,2H),5.40(m,2H),
5.30-5.24(m,4H),4.77(m,6H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,2H),3.96-
3.94(m,6H),3.80(m,2H),3.60(m,2H),3.50(m,1H),3.44-3.40(m,21H).MS(ESI):825.9
[(M-2Na)/2]-
The preparation of 6 pentasaccharides II-6 (sodium salt) of embodiment
Synthetic route:
It referring to the method for embodiment 1, is reacted by raw material of DE20 and FGH4, then through hydrogenolysis removing benzyl, generation sulphur
Pentasaccharides II-6 (sodium salt) is made in acidification reaction, hydrolysis.
Compound DEFGH60-1:1H NMR(300MHz,CDCl3)δ8.05-7.55(m,5H),7.34-7.29(m,40H),
5.99(d,2H),5.60(m,3H),5.17(m,2H),4.64–4.62(m,18H),4.23-4.19(m,4H),4.00(m,3H),
3.80-3.77(m,6H),3.70(s,6H),3.61(d,3H),3.50(d,3H),3.41-3.40(m,15H),3.36(m,3H)
.MS(ESI):1843.8[M+Na]+
Compound DEFGH61-1:1H NMR(300MHz,CDCl3) δ 8.05-7.55 (m, 5H), 5.99 (d, J=3.6Hz,
2H),5.40(m,3H),5.17(m,3.4Hz,2H),4.77(m,3H),4.71(d,2H),4.64–4.62(m,2H),4.23-
4.19(m,4H),4.10(m,1H),3.94-3.80(m,10H),3.70(d,6H),3.57-3.50(m,8H),3.41-3.40
(m,15H),3.30(m,2H),2.02(d,3H).MS(ESI):1123.4[M+Na]+
Compound DEFGH62-1:1H NMR(300MHz,CDCl3) δ 8.04-7.55 (m, 5H), 5.99 (d, J=3.6Hz,
2H),5.54(m,3H),5.24-5.17(m,7H),4.63(m,2H),4.23–4.19(m,7H),3.96-3.94(m,6H),
3.80(m,2H),3.74-3.70(m,7H),3.50-3.41(m,18H),2.02(s,3H).MS(ESI):934.9[(M-2Na)/
2]-
Pentasaccharides II-6 (sodium salt):1H NMR(300MHz,CDCl3)δ5.54(m,3H),5.40(m,2H),5.30-5.24(m,
5H),4.77(m,6H),4.64–4.62(m,2H),4.23-4.21(m,5H),4.10(m,2H),3.96-3.94(m,6H),
3.80(m,2H),3.60(m,2H),3.50-3.40(m,18H).MS(ESI):846.9[(M-2Na)/2]-
7 biological experiment of embodiment
Five sugar compounds of the invention biological activity test can using well known to a person skilled in the art method into
Row measurement.It is understood that following test methods are not limited in any way the scope of the present invention.
1.AT III combination KDThe measurement of value
Using Fluorescence Method, Perkin Elmer LS-50 type sepectrophotofluorometer is used；Excite λ 280nm, transmitting
λ338nm；Constant temperature sample room is equipped with to continuously stir at 37 DEG C.2mL Tris-HCl buffer is added in five sugar compounds
(0.01M, pH value 7,0.15M NaCl and 5-60nM people AT- III) test tube.
The ratio of III-five saccharide complex of AT- is reacted with the calculating of concentration by 1: 1 to measure, dissociation constant KDMeasurement adopt
It is analyzed with Scatchard.As a result average value ± SEM, n=3.
2. the measurement of anticoagulin xa activity
Five sugar compounds of the invention inhibit factor Xa by activation antithrombase AT III.Anti-Xa activity measurement
Using anti-Xa Amidolytic AT III method (i.e. chromogenic substrate method).At 37 DEG C, factor Xa is (in 20mM Tris/ maleic acid
In salt buffer, concentration is 7.5nkat/ milliliters, pH=7.4, NaCl 150mM；100 μ L), AT III is (in 20mM Tris/ horse
Come in phthalate buffer, concentration is 0.5 units per ml, pH=7.4, NaCl 150mM；100 μ L) and pentasaccharides of the invention (
In 20mM Tris/ maleate buffers, concentration is 0.5 units per ml, pH=7.4, NaCl 150mM；100 μ L) culture 2
Minute.Then S-2222 culture medium (Bz- lle-Glu-glycine-arginine pNA is added；1mM is in 50mM Tris-
In HCl buffer, pH=8.4,175mM NaCl, EDTA 27.5mM；100 μ L) measure remaining factor Xa.2 minutes
The acetic acid aqueous solution (100 μ L) that 50% is added afterwards stops reaction, and absorbance is measured at 405nm.Then the percentage of inhibition is calculated
Than formula: inhibiting %=100 × (A of control buffer405The A of sample405The A of)/control buffer405；With the mark calibrated
Standard relatively, uses the activity of 2015 software of Excel measurement compound.It as a result is average value ± SEM, n=3.
3. half-life period (T1/2) measurement
After intravenous administration, the pharmacokinetics of five sugar compounds of the invention is carried out in male Wistar rat
Research, dosage 100nmol/Kg.Blood sample is acquired at multiple time points after administration, by the blood sample of 9 volumes and 1 volume
The mixing of 0.129M sodium citrate solution and immediately ice bath cooling make sample receive 3000Xg at low temperature and are centrifuged 10 minutes, and-
It is frozen at 20 DEG C.The concentration of compound in every milliliter of blood plasma is measured by coagulation factor xa activity.For each compound, by dense
It spends the curve relative to the time and removes half-life period to calculate.
As a result as shown in the table:
| Compound | AT III combination KDIt is worth (nM) | Anti-Xa factor activity (units/mg) | Half-life period (T1/2,h) |
| II-1 | 24±3 | 1300±32 | 1.4±0.1 |
| II-2 | 23±2 | 950±25 | 1.3±0.1 |
| II-3 | 21±2 | 900±27 | 1.4±0.1 |
| II-4 | 21±3 | 870±24 | 1.2.±0.1 |
| II-5 | 26±2 | 1200±41 | 1.3±0.1 |
| II-6 | 25±2 | 880±21 | 1.5±0.1 |
| Fondaparinux sodium | 57±3 | 850±26 | 0.7±0.1 |
Upper table data show that five sugar compounds of the invention have potent Anti-Xa activity, compared with Fondaparinux sodium, this
The Anti-Xa activity of invention compound is its 1~1.5 times；And the half-life period of five sugar compounds of the invention is more compared with Fondaparinux sodium
It is long.
4. bleeding influences test
Male Wistar rat (weight 18-22g) is randomly divided into 6 test groups, blank group and control group, every group 12
Only.6 test groups are injected intravenously pentasaccharides II-1, II-2, II-3, II-4, II-5 and II-6, control group intravenous injection respectively
Fondaparinux sodium, blank group are injected intravenously physiological saline.Every group is further divided into four groups, respectively with 1mg/Kg, 3mg/Kg,
The dosage of 10mg/kg and 15mg/kg is administered, and five sugar compounds of the invention are in terms of its sodium salt.After 1h is administered, by the tail of rat
It cuts about 5mm and immerses in 40mL distilled water, constant temperature stirs 90min at 37 DEG C.Using blood in spectrophotometric method measurement water
The amount of Lactoferrin, Detection wavelength 540nm prepare standard curve, calculate amount of bleeding.Results are averaged ± SD, n=3.
For upper table the results show that under high dosage, the influence of five sugar compounds of the invention to hemorrhagic tendency is significant
Less than positive control Fondaparinux sodium.
Claims (17)
1. five sugar compounds of synthesis of ionic species shown in Formula II or its acid or its salt,
Wherein, R1Selected from H, SO3 -Or C1~C4 alkyl；
R2Selected from H or SO3 -。
2. five sugar compounds of synthesis of ionic species according to claim 1 or its acid or its salt, which is characterized in that be selected from
With flowering structure:
Wherein, R1For C1~C4 alkyl.
3. five sugar compounds of synthesis of ionic species according to claim 1 or its acid or its salt, which is characterized in that be selected from
With flowering structure:
Wherein, R1For C1~C4 alkyl.
4. five sugar compounds according to claim 2 or 3 or its acid or its salt, which is characterized in that R1For methyl.
5. five sugar compounds according to claim 1-3 or its acid or its salt, which is characterized in that the salt choosing
From sodium salt, sylvite, calcium salt or ammonium salt.
6. the method for preparing five sugar compounds as claimed in claim 2 or its acid or its salt, which is characterized in that include following step
It is rapid:
(1) disaccharides shown in formula DE and trisaccharide shown in formula FGH5 are reacted, preparation formula DEFGH0,
Wherein, RaFor Ac, Bn, C1~C4 alkyl；
(2) formula DEFGH0 removing benzyl is prepared into DEFGH1,
Wherein, RbFor Ac, H, C1~C4 alkyl；
(3) sulfating reaction is occurred into for compound shown in formula DEFGH1 and prepares DEFGH2,
Wherein, RcFor Ac, SO3 -, C1~C4 alkyl；
(4) by compound hydrolysis preparation formula II-1~II-3 shown in formula DEFGH2.
7. the method for preparing five sugar compounds as claimed in claim 3 or its acid or its salt, which is characterized in that include following step
It is rapid:
(1) disaccharides shown in formula DE and trisaccharide shown in formula FGH4 are reacted, preparation formula DEFGH0-1,
Wherein, RaFor Ac, Bn, C1~C4 alkyl；
(2) compound shown in formula DEFGH0-1 is removed into benzyl preparation formula DEFGH1-1,
Wherein, RbFor Ac, H, C1~C4 alkyl；
(3) by formula DEFGH1-1 occur sulfating reaction, preparation formula DEFGH2-1,
Wherein, RcFor Ac, SO3 -, C1~C4 alkyl；
(4) hydrolysis is occurred into for compound shown in formula DEFGH2-1, prepares pentasaccharides II-4~II-6.
8. the compound being shown below
Wherein, RaFor Ac, Bn, C1~C4 alkyl.
9. the compound being shown below:
10. the compound being shown below:
Wherein, RaFor Ac, Bn, C1~C4 alkyl.
11. the compound being shown below:
Wherein, RbFor Ac, H, C1~C4 alkyl.
12. the compound being shown below:
Wherein, RcFor Ac, SO3 -, C1~C4 alkyl.
13. pharmaceutical composition, it includes as active constituent compound according to any one of claims 1 to 5, its acid or
Its salt and optional pharmaceutically acceptable auxiliary material.
14. pharmaceutical composition according to claim 13, the active constituent containing 0.1mg~100mg.
15. described in compound according to any one of claims 1 to 5, its acid or any one of its salt or claim 13-14
Pharmaceutical composition in preparing drug for preventing and/or treating disease relevant to blood coagulation disorder and morbid state
Purposes, the disease and morbid state are preferably Deep vain thrombosis, thrombophlebitis, are drawn by thrombosis or embolism
Artery occlusion, Postoperative Intravenous thrombosis or embolism, pulmonary embolism and the non-ST elevation acute myocardial infraction risen.
16. the method for preventing and/or treating relevant to blood coagulation disorder disease and morbid state comprising Xiang Youxu
The individual wanted gives compound according to any one of claims 1 to 5, any in its acid or its salt or claim 13-14
Pharmaceutical composition described in, the disease and morbid state are preferably Deep vain thrombosis, thrombophlebitis, by thrombus
Artery occlusion caused by formation or embolism, Postoperative Intravenous thrombosis or embolism, pulmonary embolism and non-ST elevation acute myocardial infraction.
17. appointing for preventing and/or treating in disease relevant to blood coagulation disorder and the Claims 1 to 5 of morbid state
Compound described in one, its acid or any one of its salt or claim 13-14 described in pharmaceutical composition, the disease and
Morbid state is preferably Deep vain thrombosis, thrombophlebitis, the artery occlusion as caused by thrombosis or embolism, postoperative
Venous thronbosis or embolism, pulmonary embolism and non-ST elevation acute myocardial infraction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020114418A1 (en) * | 2018-12-05 | 2020-06-11 | 南京正大天晴制药有限公司 | Anticoagulant pentasaccharide compound, composition, and preparation method and medical use thereof |
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| CN1169433A (en) * | 1996-05-08 | 1998-01-07 | 阿克佐诺贝尔公司 | Carbohydrate derivatives |
| CN1222524A (en) * | 1997-11-19 | 1999-07-14 | 阿克佐诺贝尔公司 | Carbohydrate derivatives |
| CN1228785A (en) * | 1996-07-19 | 1999-09-15 | 萨诺费公司 | Synthetic polysaccharides, preparation method therefor and pharmaceutical compositions containing same |
| CN103145774A (en) * | 2013-03-21 | 2013-06-12 | 苏州鸿洋医药科技有限公司 | Anticoagulation pentose and preparation method thereof |
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| US5543403A (en) * | 1990-04-23 | 1996-08-06 | Akzo Nobel Nv | Sulfated glycosaminoglycanoid derivatives of the heparin and heparan sulfate type |
| CN1169433A (en) * | 1996-05-08 | 1998-01-07 | 阿克佐诺贝尔公司 | Carbohydrate derivatives |
| CN1228785A (en) * | 1996-07-19 | 1999-09-15 | 萨诺费公司 | Synthetic polysaccharides, preparation method therefor and pharmaceutical compositions containing same |
| CN1222524A (en) * | 1997-11-19 | 1999-07-14 | 阿克佐诺贝尔公司 | Carbohydrate derivatives |
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