HK1197915B - Sulfated oligosaccharide derivatives - Google Patents

Description

Sulfated oligosaccharide derivatives

The application is a divisional application of Chinese patent application No. 200580006833.8 (PCT/AU 2005/000314) with the application date of 3/4/2005 entitled "sulfated oligosaccharide derivatives".

Technical Field

The invention described herein relates to compounds having activity as inhibitors of heparan sulfate-binding proteins and heparanase. In particular, the invention relates to sulfated oligosaccharide derivatives, although the scope of the invention is not necessarily limited thereto. In particular, the present invention relates to multiply sulfated oligosaccharide derivatives, preferably derivatized at C-1 at the reducing end and/or at C-6 at the non-reducing end of the monosaccharide unit. The invention also relates to methods of preparing such compounds, compositions comprising such compounds, and the use of such compounds and compositions thereof for the treatment of antiangiogenic, antimetastatic, anti-inflammatory, antimicrobial, anticoagulant and/or antithrombotic in mammalian subjects. The compounds and compositions thereof also have the effect of lowering blood triglyceride levels and inhibiting cardiovascular disease in a mammalian subject. The compounds also have an effect on the prevention of the above-mentioned conditions when administered to a mammalian subject.

Background

The known sulfated oligosaccharide reagent PI-88[1,2] (see Compound 1 below) has proven to be a valuable tumor growth and metastasis inhibitor [1,3] and is undergoing phase II clinical trials in cancer patients [4 ]. PI-88 exerts an anti-angiogenic effect by inhibiting the interaction of angiogenic growth factors (in particular FGF-1, FGF-2 and VEGF) and their receptors with heparan sulfate [1,5 ]. In addition, PI-88 is a potent inhibitor of heparanase, a glycosidase that cleaves the heparan sulfate side chain of proteoglycans, a major component of the extracellular matrix (ECM) and basement membrane surrounding tumor cells [1,2 ]. Heparanase has been closely linked to blood vessels: it is capable of releasing active heparan sulfate-bound angiogenic growth factors from the ECM and is associated with degradation of the ECM and subsequent tissue remodeling associated with the growth of new blood vessels [6 ]. The degradation of the ECM by heparanase is also crucial in the spread (metastasis) of the tumour cells by allowing them to enter the bloodstream and to settle at a distance where they are able to form secondary tumours [6,7 ].

In addition to its anti-angiogenic effect, PI-88 inhibits the coagulation system by: (i) inhibition of proteases in the intrinsic pathway, (II) stimulation of Tissue Factor Pathway Inhibitor (TFPI) release, and (iii) activated heparin cofactor II-mediated inhibition of thrombin. However, PI-88 does not interact with AT III and thus shows no anti-Xa or ATIII-mediated anti-IIa activity [8,9 ]. In vivo studies in monkeys have shown that low doses of PI-88 promote the release of fully heparan sulfate-bound TFPI from the vascular cell wall [9 ]. In addition to its effects on blood coagulation, TFPI has recently been shown to be an anti-angiogenic agent [10] and a metastasis inhibitor [11 ]. PI-88 has also been shown to block vascular smooth muscle cell proliferation and intimal thickening [12], inhibit Herpes Simplex Virus (HSV) infection of cells and cell-to-cell spread of HSV-1 and HSV-2 [13], and inhibit proteinuria in passive glomerulonephritis [14 ].

PI-88 is a mixture of monophosphorylated manno oligosaccharides with a high degree of sulfation, ranging in size from di-to hexoses [15,16 ]. PI-88 was prepared by complete sulfonation [2,16] of the oligosaccharide phosphate moiety (2) (see formula I after this paragraph), which oligosaccharide phosphate moiety (2) was obtained by mild, acid-catalyzed hydrolysis of the extracellular phosphomannan of the yeast Pichia (Hansenula) holstii NRRL Y-2448 [17,18 ]. The major components are penta-and tetrasaccharide phosphates 3 (-60%) and 4 (-30%), respectively, while the remaining 10% are composed of di-, tri-and hexose phosphates (5-7) and tetrasaccharides (not shown) [15,16 ].

Formula I

Various other multiply sulfated oligo-and polysaccharides and their derivatives are known to exhibit a similar type of biological activity to PI-88[ 19-25 ]. These biological activities are attributed to the inhibition of various Heparan Sulfate (HS) -binding proteins. It is an object of the present invention to produce derivatives of PI-88 that have similar biological activity but improved properties, e.g. in their pharmacokinetics and/or ADME (absorption, distribution, metabolism, excretion) aspects. It is a further object of the present invention to provide compounds containing a single carbon skeleton to facilitate their synthesis and characterization.

Summary of The Invention

According to a first embodiment of the present invention, there is provided a compound of formula (la):

X－[Y]_n－Z－UR¹

II

wherein;

x, Y and Z are each monosaccharide units having groups UR bound by single or multiple bonds to each of the non-linked carbons of X, Y and Z, except having groups UR bound by single or multiple bonds^lOther than carbon-1 of monosaccharide Z;

n is an integer of 0 to 6;

each U is independently C, N, S or O or their higher oxidation state, including CO, COO, NO₂、S(O)、S(O)O；

Each R is independently SO₃M or H, wherein M is any pharmaceutically acceptable saltAcceptable cations are either any alkyl, aryl, acyl, aroyl, alkylsulfonyl, arylsulfonyl, PEG derivative, H, or group

Wherein independently in each AB group, a is O or NH and B is H or M, wherein M is as described above, or alkyl, aryl or any other suitable group;

R^lis SO₃M, H, alkyl, aryl, acyl, aroyl, alkylsulfonyl, arylsulfonyl, PEG or PEG derivatives, or R^lTogether with U being N₃Or a substituted triazole or derivative, or a substituted tetrazole or derivative, or a substituted aryl or derivative, or a substituted heteroaryl or derivative;

provided that when all URs are present^lAnd UR group is OSO₃M or OH (other than the exocyclic methylene group of said monosaccharide X), the exocyclic methylene group of said monosaccharide X cannot be OPO₃M₂A group.

According to a second embodiment of the present invention there is provided a pharmaceutical or veterinary composition for the prevention or treatment of a condition resulting from angiogenesis, metastasis, inflammation, coagulation/thrombosis, elevated blood triglyceride levels, microbial infection and/or cardiovascular disease in a mammalian subject, the composition comprising at least one compound according to the first embodiment together with a pharmaceutically or veterinarily acceptable carrier or diluent for said at least one compound.

A third embodiment of the invention comprises the use of a compound according to the first embodiment for the manufacture of a medicament for the prevention or treatment of a condition resulting from angiogenesis, metastasis, inflammation, coagulation/thrombosis, elevated blood triglyceride levels, microbial infection and/or cardiovascular disease in a mammalian subject.

According to a fourth embodiment of the present invention there is provided a method for preventing or treating a condition resulting from angiogenesis, metastasis, inflammation, coagulation/thrombosis, elevated blood triglyceride levels, microbial infection and/or cardiovascular disease in a mammalian subject, the method comprising administering to said subject an effective amount of at least one compound according to the first embodiment or a composition comprising said at least one compound.

Further embodiments of the invention include novel intermediates and synthetic routes in the preparation of the sulfated oligosaccharides of the first embodiment.

Preferred compounds according to the invention, wherein the monosaccharide molecule is a dedicated D-mannose and the glycosidic linkages are α - (l → 2) and α - (l → 3), are described in the following structures:

r, R therein^lU and n are as described above.

In order that the invention may be more readily understood and put into practical effect, one or more preferred embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings.

Brief Description of Drawings

FIG. 1 shows the effect of PI-88 like compounds on HSV-1 infectivity (A) and HSV-1 cell-to-cell transmission (B). In panel a, the results are expressed as the number of viral Plaque Forming Units (PFU) formed in cells infected with the compound-treated virions relative to the pseudo-treated control. In panel B, the results are expressed as the average area of 20 viral plaques formed in the continuous presence of the compound relative to the percentage of pseudo-treated control cells.

Description of The Preferred Embodiment

The present inventors have found that a number of sulfated oligosaccharide derivatives can be synthesized using a number of different strategies as broadly described below and exemplified in the examples. These compounds have utility in preventing or treating a disorder resulting from angiogenesis, metastasis, inflammation, coagulation, thrombosis, elevated blood triglyceride levels, microbial infection, and/or cardiovascular disease in a mammalian subject. This effect results from the ability of the compounds to block the binding of heparan sulfate-binding proteins to their receptors, or to inhibit the activity of the heparanase.

With respect to the subject compounds of formula II, the monosaccharide units X, Y and Z can be, for example, any hexose or pentose and can be D or L isomers. Such hexoses include glucose, mannose, altrose, allose, talose, galactose, idose, and gulose. Such pentoses include ribose, arabinose, xylose, and lyxose. The glycosidic linkages of the monosaccharide units may be of only one type or of different types, depending on structure and linkage.

The pharmaceutically acceptable cation M is preferably sodium.

With respect to the integer n, a preferred value is 3, thereby providing the pentasaccharide compound.

Preferred suitable R^lThe group is n-octyl.

Said anomeric configuration, where applicable, being in UR of the compound of formula II¹It may be α or β or an anomeric α/β mixture.

With respect to said substituents in the definitions of the compounds of formula II given above, the term "alkyl", when used alone or in compound words such as "arylalkyl", refers to a straight, branched or cyclic hydrocarbon group, preferably C_1-20Example ofSuch as C_1-10. For example, the term "C_l-C₆Alkyl "refers to a straight, branched, or cyclic alkyl of 1 to 6 carbon atoms. "C_l-6Examples of "alkyl" include methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2-dimethylbutyl, 3-methylpentyl and 2, 3-dimethylpropyl. Ring C_1-6Examples of alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Other examples of alkyl groups include: heptyl, 5-methylhexyl, 1-methylhexyl, 2-dimethylpentyl, 3, 3-dimethylpentyl, 4-dimethylpentyl, 1, 2-dimethylpentyl, 1, 3-dimethylpentyl, 1, 4-dimethyl-pentyl, 1,2, 3-trimethylbutyl, 1, 2-trimethylbutyl, 1, 3-trimethylbutyl, octyl, 6-methylheptyl, 1,3, 3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6-or 7-methyl-octyl, 1-, 2-, 3-, 4-or 5-ethylheptyl, 1-, 2-or 3-propylhexyl, 2, 3-dimethylpentyl, 1, 3-trimethylbutyl, 1,2, 3-trimethylbutyl, 1, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-and 8-methylnonyl, 1-, 2-, 3-, 4-, 5-or 6-ethyloctyl, 1-, 2-, 3-or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6-or 7-ethylnonyl, 1-, 2-, 3-, 4-or 5-propyloctyl, 1-, 2-or 3-butylheptyl, 1-pentylhexyl, dodecyl, and the like, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5-or 6-propylnonyl, 1-, 2-, 3-or 4-butyloctyl, 1-2-pentylheptyl, and the like. Alkyl groups may be optionally substituted by one or more optional substituents as defined herein. Optionally, the linear, branched or cyclic hydrocarbon group (having at least 2 carbon atoms) may contain one, two or more unsaturations to form an alkenyl or alkynyl group, preferably C_2-20Alkenyl, more preferably C_2-6Alkenyl, or C_2-20Alkynyl, more preferably C_2-6Alkynyl. Examples thereof include compounds containing one or two or more double bonds, or one or moreA hydrocarbon residue of two or more triple bonds. Thus, "alkyl" is considered to include alkenyl and alkynyl groups.

The term "aryl", when used alone or in compound words such as "arylalkyl", denotes a single, polynuclear, conjugated or fused residue of an aromatic hydrocarbon or aromatic heterocyclic (heteroaryl) ring system, wherein one or more carbon atoms of the cyclic hydrocarbon residue is substituted with a heteroatom to provide an aromatic residue. In the case where two or more carbon atoms are replaced, this may be by two or more of the same heteroatoms or by different heteroatoms. Suitable heteroatoms include O, N, S and Se.

Examples of "aryl" include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl, and the like. Preferred hydrocarbon aryl groups include phenyl and naphthyl. Preferred heterocyclic aryl groups include pyridyl, thienyl, furyl, pyrrolyl. The aryl group may be optionally substituted with one or more optional substituents as defined herein.

The term "acyl" refers to the group-C (O) -R, where R is an alkyl or aryl group. Examples of the acyl group include straight-chain or branched alkanoyl groups such as acetyl, propionyl, butyryl, 2-methylpropionyl, pentanoyl, 2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and eicosanoyl; cycloalkylcarbonyl groups such as cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl groups such as phenylalkanoyl groups (e.g., phenylacetyl, phenylpropionyl, phenylbutyryl, phenylisobutyryl, phenylpentanoyl, and phenylhexanoyl) and naphthylalkanoyl groups (e.g., naphthylacetyl, naphthylpropionyl, and naphthylbutyryl). Since the R group may be optionally substituted as described above, "acyl" is considered to mean an optionally substituted acyl group.

Optional substituents for alkyl, aryl or acyl include halogen (bromo, fluoro, chloro, iodo), hydroxy, C_1-6Alkyl (e.g., methyl, ethyl, propyl (n-and iso-isomers)), C_l-6Alkoxy (e.g., methoxy, ethoxy, propoxy (n-and iso-isomers), butoxy (n-, sec-and tert-isomers), nitro, amino, C_1-6Alkylamino (e.g., methylamino, ethylamino, propyl (n-and iso-isomer) amino), C_1-6Dialkylamino (e.g., dimethylamino, diethylamino, diisopropylamino), halomethyl (e.g., trifluoromethyl, tribromomethyl, trichloromethyl), halomethoxy (e.g., trifluoromethoxy, tribromomethoxy, trichloromethoxy), and acetyl.

The 5-6 membered heterocyclic group includes aromatic 5-6 membered heterocyclic groups (heteroaryl groups) as described above and non-aromatic 5-6-membered heterocyclic groups containing one or more heteroatoms (preferably 1 or 2) independently selected from O, N, S and Se. Examples thereof include dioxane, pyranyl, tetrahydrofuranyl, piperidinyl, morpholino, piperazinyl, thiomorpholino, and sugars.

The degree of sulfation of the compounds according to the invention is generally at least 50%. That is, at least 50% of the R groups of the oligosaccharide derivative contain SO₃And M. The degree of sulfation is generally 70 to 100% and preferably at least as high as 90%.

The PI-88 derivatives of formula II can be prepared by stepwise synthetic routes or by starting from the PI-88 backbone already present in situ (using the readily available compounds 8-11; see formula I above) and making the required modifications thereto. The inventors have determined from consideration of the structure of PI-88(1) and its precursor (2) that there are two preferred derivatization sites: the reducing end (A) and the terminal 6-position on the non-reducing end (B) are shown in the following structural formula.

R=SO₃Na or H, R¹=PO₃Na₂,n=0-4

It should be noted that all di-, tri-, tetra-and pentasaccharide (and larger) derivatives can be prepared by the same chemical process. However, the pentasaccharide derivatives are preferred because they are the most biologically active [1,2,5,8,13 ]. All of the resulting derivatives are then deprotected (typically deacetylated with NaOMe) and the resulting polyhydroxy compounds are sulfonated with a sulfonating agent such as sulfur trioxide pyridine complex or sulfur trioxide trimethylamine complex.

As indicated above, the compounds according to the invention have a role in the prevention or treatment of a condition resulting from angiogenesis, metastasis, inflammation, coagulation, thrombosis, elevated blood triglyceride levels, microbial infection or cardiovascular disease in a mammalian subject. The compounds have particular utility in the treatment of the above-mentioned conditions in humans. Generally, the compounds are administered as components of pharmaceutical compositions described in the following paragraphs. As will be exemplified below, the compounds show similar or better activity than PI-88 itself.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may include solid carriers such as gums or adjuvants or inert diluents. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oils, or synthetic oils. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. Such compositions and formulations will generally comprise at least 0.1% by weight of the compound.

Parenteral administration includes administration by the following routes: intravenously, dermally or subcutaneously, nasally, intramuscularly, intraocularly, transepithelially, intraperitoneally, and topically. Topical administration includes dermal, ocular, rectal, nasal administration, and administration by inhalation or by aerosol means. For intravenous, cutaneous or subcutaneous injection, or injection at a site in need of treatment, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art will be well able to prepare suitable solutions for use, for example, solutions of the subject compounds or derivatives thereof.

In addition to at least one of the compounds and the carrier or diluent, the composition according to the invention may further comprise pharmaceutically or veterinarily acceptable excipients, buffers, stabilizers, isotonicizing agents, preservatives or antioxidants or any other material known to the person skilled in the art. The skilled artisan will appreciate that such materials should be non-toxic and will not interfere with the efficacy of the compounds. The exact nature of any additives may depend on the route of administration of the composition: i.e., whether the composition is to be administered orally or parenterally. With respect to buffers, aqueous compositions generally include materials such that the composition is maintained at a near physiological pH or at least within a range of about pH5.0 to 8.0.

The composition according to the invention may also comprise active ingredients other than at least one of said compounds. Such ingredients will be selected primarily for their efficacy as anti-angiogenic, anti-metastatic, anti-inflammatory, anti-coagulant, anti-microbial and anti-thrombotic agents, as well as agents effective against elevated blood triglyceride levels and cardiovascular disease, but their efficacy may be selected according to any relevant condition.

The pharmaceutical or veterinary composition according to the invention will be administered to the subject in a prophylactically or therapeutically effective amount as necessary for the particular situation under consideration. The actual amount of the at least one compound administered in a composition, as well as the frequency and number of administrations — the course of treatment, will depend on the nature and severity of the condition being treated or the desired prevention. Prescription of treatment, e.g., determination of dosage, etc., will be within the skill of the practitioner or veterinarian responsible for caring for the subject. Generally, however, a composition for administration to a human subject will comprise from about 0.01 to 100mg of the compound per kg body weight and more preferably from about 0.1 to 10mg/kg body weight.

The compounds may be included in the compositions as pharmaceutically or veterinarily acceptable derivatives. As used herein, "derivatives" of said compounds include salts, and metal ions such as Mn²⁺And Zn²⁺Such as in vivo hydrolysable esters, free acids or bases, hydrates or prodrugs. Compounds having acidic groups such as phosphates or sulfates can form salts with alkali or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and tris (2-hydroxyethyl) amine. Salts may also be formed between a compound and a basic group, for example an amine, and an inorganic acid such as hydrochloric acid, phosphoric acid or sulfuric acid, or an organic acid such as acetic acid, citric acid, benzoic acid, fumaric acid or tartaric acid. Compounds having both acidic and basic groups can form internal salts.

Esters may be formed between the hydroxyl or carboxylic acid groups present in the compound and the appropriate carboxylic acid or alcohol reactive partner using techniques well known to those skilled in the art.

Prodrug derivatives of the compounds of the invention may be converted to the parent compound in vivo or in vitro. In general, at least one biological activity of the parent compound may be inhibited in prodrug forms of the compound, and the prodrug may be activated by conversion to the parent compound or a metabolite thereof. Examples of prodrugs are glycolipid derivatives, wherein one or more lipid moieties are provided as substituents on said moiety, which are cleaved by an enzyme having phospholipase activity to release the free form of the compound. Prodrugs of the compounds of the present invention include the use of protecting groups which may be removed in vivo to release the active compound or to inhibit the clearance of the drug. Suitable protecting groups are known to those skilled in the art and include acetate groups.

As also indicated above, the compounds according to the invention have an effect in the manufacture of a medicament for the prevention or treatment of a condition resulting from angiogenesis, metastasis, inflammation, coagulation/thrombosis, microbial infection, elevated blood triglyceride levels and/or cardiovascular disease in a mammalian subject. Methods for preparing such medicaments are known to the person skilled in the art and include methods for preparing pharmaceutical compositions as described above.

A general description will now be given of a synthetic route to the compounds according to the invention. For simplicity, R is shown in all subsequent figures, drawings and tables^lMan to represent α - (l → 3) -ligation₄Tetrasaccharide moieties (with or without terminal 6-O-phosphate groups) unless otherwise indicated.

General procedure

Glycoside derivatives (O-, S-and C-glycosides) of PI-88

Glycoside derivatives can be readily prepared by activating the oligosaccharide for saccharification (with or without terminal 6-O-phosphate groups) and condensing it with the appropriate alcohol. Suitable processes are Lewis acid catalyzed or promoted reactions of peracetylated saccharides, e.g., 12, with alcohol acceptors, e.g., to give 13 and 14. Where a more inert acceptor is required, a more reactive glycosyl donor needs to be prepared, e.g., the trichloroacetimidate 15 is used to prepare pegylated derivatives 16 and 17 (table 1).

Chart 1

Various other types of donors are known in the art and are suitable as donors, for example, thioglycosides, halides, n-pentenyl glycosides, selenoglycosides, and the like. Those skilled in the art will recognize that S-and C-glycosides can be prepared by similar or related methods known in the literature, for example by using the appropriate thiol (or thiol derivative) or a known carbon nucleophile having a suitable activated donor (e.g., allyltrimethoxysilane or a suitable phenol). The product can then be easily deacetylated and sulfonated. The product of the saccharification may be a single anomer (alpha or beta) or a mixture of two anomers. Both the pure alpha and beta anomers and anomer mixtures are suitable for subsequent conversions. This also applies to other derivatives obtained by manipulation of the anomeric centers as described in the subsequent section. Thus, where a single anomer is indicated, this means that the opposite anomer or a mixture of both anomers is also required. The initially formed glycoside may be further derivatized, depending on the nature of the aglycone, as will also be clear to those skilled in the art. As an example, if one uses 2-bromohexanol as the alcohol, the product can be converted to an azide (18). This is an extremely variable compound (scheme 2) and can be further functionalized by, for example, cycloaddition with a compound containing a suitable dipolar affinity. Alternatively, the azide may be reduced to an amine and then further functionalized, for example, by alkylation, acylation, 4-component Ugi condensation, and the like.

Chart 2

N-linked derivatives

From 12, with TMSN₃To give the azide 19 (mainly α)) Alternatively, α -bromide may be initially formed and then NaN used₃Substitution to form exclusively β -azide 20 (scheme 3.) for example, the bromide may also be used as an intermediate in the preparation of thioglycosides or isothiocyanates the azide may be deprotected and sulfonated, or reduced and acylated with various acid chlorides to provide a series of glycosyl amides (scheme 3).

Chart 3

Non-reducing terminal derivatives

Derivatization can also be effected at the non-reducing end, for example by using phosphorylated oligosaccharides (individually or as a mixture) and by derivatization of the phosphate group, for example to prepare phosphates or phosphoramides. In practice, suitable compounds can be prepared as the reducing end is also derivatized with similar or different functional groups.

Having broadly described the invention, non-limiting examples of the compounds, their synthesis, and their biological activity will now be given.

Examples

Neutral mannan-oligosaccharide

(a) Manno-oligosaccharides were isolated by size exclusion chromatography from the neutral fraction of weak acid catalyzed hydrolysis of extracellular phosphomannan from P.holstii NRRL Y-2448 according to literature procedure [17] (8) α -D-Man- (1 → 2) -D-Man, (9) α -D-Man- (l → 3) - α -D-Man- (1 → 2) -D-Man, (10) α -D-Man- (1 → 3) - α -D-Man- (1 → 3) - α -D-Man- (1 → 2) -D-Man and (11) α -D-Man- (l → 3) - α -D-Man- (l → 3) - α -D-Man- (1 → 3) - α -D-Man- (1 → 2) -D-Man. Alternatively, the oligosaccharides 8-11 were synthesized in a stepwise manner from monosaccharide building blocks (see below) as described in example 1.

(b) Alternatively, the neutral moiety is acetylated directly (excess Ac)₂O/pyridine) and the single peracetylated oligosaccharide was isolated by flash chromatography (silica gel) and used directly in this form for the next step.

(c) In another process, the peracetylated mixture from (b) is used directly in the next step and the individual products are then isolated by flash chromatography.

General procedure for deacetylation

A solution of the peracetate in dry methanol (0.1M) was treated with a solution of sodium methoxide in methanol (1.35M, 0.2-0.6 equiv.). The mixture was stirred at room temperature for 1-3 hours (monitored by TLC). Adding acidic resin(H⁺Form) pH =6-7, the mixture was filtered and the resin was rinsed with methanol. The combined filtrate and rinse was concentrated in vacuo and dried thoroughly to obtain the polyol product.

General procedure for sulfonation

Reacting the polyol with SO₃Trimethylamine or SO₃A mixture of pyridine complexes in DMF (2 equivalents per alcohol) was heated (60 ℃ C., o/n). The cooled (room temperature) reaction mixture was treated with MeOH and then Na was added₂CO₃(10% w/w) to make it basic (to pH)>10). The mixture is filtered and evaporated and co-evaporated (H)₂O) the filtrate. Dissolving the crude polysulfated material in H₂O and size exclusion chromatography (see below) to give the sulfated product. When desired, after freeze-drying, the product is passed through an ion exchange resin column (Na⁺Form, 1 × 4cm, deionisedH₂O, 15mL) to uniformly convert the product to the sodium salt form. The collected solution was evaporated and freeze-dried to give the final product as a colorless glass or white powder.

Size exclusion chromatography

0.1M NH was applied to Bio-Gel P-2 in a 5 × 100cm column₄ ⁺·HCO₃ ^-Size exclusion chromatography was performed at a flow rate of 2.8mL/min and fractions collected over 2.8 minutes (7.8 mL). The fractions were analyzed for carbohydrate content by spotting on silica gel plates and imaging with carbonization, and/or for multiply charged species by the dimethylmethylene blue test. Finally, by CE¹⁵The components were checked for purity and those were decanted and freeze-dried, which were considered salt-free, in the presence of by-products of inferior sulphation or other organic salt impurities (usually only in small amounts, but quite often detected), they were completely removed using LH20 column chromatography (2 × 95cm, deionized water, 1.2 mL/min, 3.5 min per vial).

EXAMPLE 1 Total Synthesis of neutral mannan-oligosaccharide (8-11) from Pichia

Benzyl 2-O- (3-O-allyl-2, 4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) -3,4, 6-tri-O-benzyl-alpha-D-mannopyranoside (24)

3-O-allyl-2, 4, 6-tri-O-benzoyl- α -D-mannopyranosyl trichloroacetimidate [26 ]](902mg, 1.21mmol) with benzyl 3,4, 6-tri-O-benzyl- α -D-mannopyranoside [27](723mg, 1.34mmol) of a mixture in 1,2-DCE (10mL) over molecular sieves (1.0g ofPowder) was stirred under argon (30 minutes). In thatThe mixture was cooled (0 ℃) with continued stirring (10 min) before TMSOTf (219. mu.L, 1.21mmol) was added. After a period of time (10 min), Et was introduced₃N (100 μ L) and the mixture was filtered. The solvent was evaporated and the residue was treated with FC (10-50% EtOAc/hexanes) to give the tribenzoate (24) as a colorless oil (1.14g, 84%).¹H NMR(CDCl₃)3.67-3.81,3.88-3.95,4.06-4.15,4.30-4.35(4m,12H;H-2^I,-3^I,-4^I,-5^I,-6a^I,-6b^I,-3^II,-5^II,-6a^II,-6b^II,OCH₂),4.94-4.70(m,7H;CH₂Ph),4.84(d,1H,J_A,B10.8Hz, A of the AB quartet), 4.93-4.96,5.04-5.09(2m,2H; = CH)₂),5.02(d,1H,J_1,21.9Hz;H-1^I),5.24(d,1H;J_1,21.9Ha;H-1^II),5.59-5.69(m,1H;=CH),5.72(dd,1H,J_2,33.1Hz;H-2^II),5.75(dd,1H,J_{3, 4}9.8,J_4,59.9Hz;H-4^II),7.09-7.58,7.97-8.06(2m,35H;Ar)。¹³C NMR(CDC1₃)61.50,63.49(2C;C-6^I,-6^II),68.63,69.17,69.31,69.46,69.64,71.08,72.04,72.64,73.60,74.73,75.30,75.38(13C;C-3^I,-4^I,-5^I,-2^II,-3^II,-4^II,-5^II,OCH₂,CH₂Ph),79.97(C-2^I),98.52,99.60(C-1^I,-1^II),117.67(=CH₂),127.70-138.43(43C;=CH,Ar),165.61,165.69,166.42(3C;C=O)。

Benzyl 2-O- (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) -3,4, 6-tri-O-benzyl-alpha-D-mannopyranoside (25)

PdCl₂(40mg) was added to a solution of the allyl ether (24) (1.09g, 0.97mmol) in MeOH (10mL) and 1,2-DCE (10mL) and the combined mixture was heated (70, 40 min). Thereafter, evaporatingThe solvent and the residue was treated with FC (20-30% EtOAc/hexanes) to give the alcohol (25) as a colorless oil (0.96g, 91%).¹HNMR(CDCl₃)3.68-3.81,3.97-4.06,4.32-4.71(3m,18H;H-2^I,-3^I,-4^I,-5^I,-6a^I,-6b^I,-3^II,-5^II,-6a^II,-6b^II,CH₂Ph),4.84(d,1H,J_A,B12Hz, A of the AB quartet), 5.05(d,1H, J)_l,21.9Hz;H-1^I),5.26(d,1H;J_1,21.9Ha;H-1^II),5.61(dd,1H,J_2,33.3Hz;H-2^II),5.67(dd,1H,J_3,49.8,J_{4, 5}9.9Hz;H-4^II),7.13-7.40,7.48-7.59,7.98-8.06(3m,35H;Ar)。¹³C NMR(CDC1₃)60.61,63.32(2C;C-6^I,-6^II),69.06,69.12,69.25,69.44,70.45,72.14,72.65,72.77,73.48,74.79,75.48,75.47,76.23(13C;C-3^I,-4^I,-5^I,-2^II,-3^II,-4^II,-5^II,OCH₂,CH₂Ph),79.66(C-2^I),98.34,99.40(C-1^I,-1^II),127.70-138.47(42C;Ar),165.97,166.36,166.97(3C;C=O)。

Benzyl 2-O- [ (3-O-allyl-2, 4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) - (1 → 3) - (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) ] -3,4, 6-tri-O-benzyl-alpha-D-mannopyranoside (26)

A mixture of 3-O-allyl-2, 4, 6-tri-O-benzoyl- α -D-mannopyranosyl trichloroacetimidate (742mg, 1.01mmol) and the alcohol (25) (908mg, 0.84mmol) in 1,2-DCE (10mL) was sieved (1.0g ofPowder) was stirred under argon (30 minutes). The mixture was stirred further before addition of TMSOTf (181. mu.L, 1.01mmol)Cooled (0 ℃ C.) (10 minutes). After a period of time (10 min), Et was introduced₃N (100 μ L) and the mixture was filtered. The solvent was evaporated and the residue was treated with FC (10-50% EtOAc/hexanes) to give the hexabenzoate (26) as a colorless oil (1.26g, 90%).¹H NMR(CDCl₃)3.51-3.56,3.66-4.06,4.23-4.27,4.30-42,4.47-4.72,4.78-4.86(6m,26H;H-2^I,-3^I,-4^I,-5^I,-6a^I,-6b^I,-3^II,-5^II,-6a^II,-6b^II,-3^III,-5^III,-6a^III,-6b^III,OCH₂,=CH₂,CH₂Ph),5.04(d,1H,J_1,21.7Hz;H-1^I),5.15(dd,1H,J_1,21.8,J_2,32.7Hz;H-2^II),5.26(d,1H;H-1^II),5.28(d,1H,J_1,21.7Hz;H-1^III),5.33-5.43(m,1H;=CH),5.77-5.82(m,2H;H-4^II,-2^III),5.92(dd,1H,J_3,49.5,J_{4, 5}9.8Hz;H-4^III),7.00-7.61,7.80-8.19(2m,50H;Ar)。

Benzyl 2-O- [ (2,4, 6-tri-O-benzoyl- α -D-mannopyranosyl) - (1 → 3) - (2,4, 6-tri-O-benzoyl- α -D-mannopyranosyl) ] -3,4, 6-tri-O-benzyl- α -D-mannopyranoside (27)

PdCl₂(40mg) was added to a solution of the allyl ether (26) (394mg, 241. mu. mol) in MeOH (10mL) and 1,2-DCE (10mL) and the combined mixture was heated (70, 60 min.). Thereafter, the solvent was evaporated and the residue was treated with FC (20-30% EtOAc/hexanes) to give the alcohol (27) as a colorless oil (317mg, 84%).¹HNMR(CDCl₃)3.67-3.82,3.91-3.99,4.01-4.21,4.29-4.71(4m,21H；H-2^I,-3^I,-4^I,-5^I,-6a^I,-6b^I,-3^II,-5^II,-6a^II,-6b^II,-3^III,-5^III,-6a^III,-6b^III,CH₂Ph),4.83(d,1H,J_A,B10.9Hz, A of the AB quartet), 5.03-5.05(m,2H; H-1)^I,-2^II),5.25-5.28(m,2H;H-1^II,-1^III),5.63(dd,1H,J_3,4=J_4,59.9Hz;H-4^II),5.77(dd,1H,J_1,22.0,J_2,33.1Hz;H-2^III),5.92(dd,1H,J_3,49.7,J_{4, 5}9.9Hz;H-4^III),6.99-7.62,7.80-8.16(2m,50H;Ar)。

Benzyl 2-O- [ (3-O-allyl-2, 4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) - (1 → 3) - (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) ] -3,4, 6-tri-O-benzyl-alpha-D-mannopyranoside (28)

A mixture of 3-O-allyl-2, 4, 6-tri-O-benzoyl- α -D-mannopyranosyl trichloroacetimidate (102mg, 138. mu. mol) and the alcohol (27) (135mg, 86.5. mu. mol) in 1,2-DCE (6mL) was placed over molecular sieves (100mg ofPowder) was stirred under argon (30 minutes). The mixture was cooled (0 °) with continued stirring (10 min) before addition of TMSOTf (25 μ L,138 μmol). After a period of time (10 min), Et was introduced₃N (100 μ L) and the mixture was filtered. The solvent was evaporated and the residue was treated with FC (10-50% EtOAc/hexanes) to give the nonabenzoate (28) as a colorless oil (173mg, 94%).¹H NMR(CDCl₃)3.44-3.49,3.60-3.99,4.05-4.16,4.42-4.44,4.48-4.68,4.73-4.77(6m,30H;H-2^I,-3^I,-4^I,-5^I,-6a^I,-6b^I,-3^II,-5^II,-6a^II,-6b^II,-3^III,-5^III,-6a^III,-6b^III,-3^IV,-5^IV,-6a^IV,-6b^IV,OCH₂,=CH₂,CH₂Ph),4.83(d,1H,J_A,B10.9Hz, A of the AB quartet), 5.01-5.04(m,2H; H-1)^I,-2^III),5.19-5.23(m,1H;H-2^II),5.27-5.40(m,4H;H-1^I,-1^II,-1^III,=CH₂),5.61(dd,1H,J_3,4=_4,59.9Hz;H-4^Ⅳ),5.77(dd,1H,J_1,22.0,J_2,33.1Hz;H-2^IV),5.90-5.96(m,2H;H-4^II,-4^III),7.01-7.56,770-8.16(2m,65H;Ar)。

Benzyl 2-O- [ (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) - (1 → 3) - (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) ] -3,4, 6-tri-O-benzyl-alpha-D-mannopyranoside (29)

PdCl₂(30mg) was added to a solution of the allyl ether (28) (155mg, 70.4. mu. mol) in MeOH (5mL) and 1,2-DCE (5mL) and the combined mixture was heated (70, 40 min.). Thereafter, the solvent was evaporated and the residue was subjected to FC (20-40% EtOAc/hexanes) to give the alcohol (29) as a colorless oil (97mg, 64%).¹H NMR(CDC1₃)3.67-3.82,3.90-4.10,4.24-4.68(3m,26H;H-2^I,-3^I,-4^I,-5^I,-6a^I,-6b^I,-3^II,-5^II,-6a^II,-6b^II,-3^III,-5^III,-6a^III,-6b^III,-3^IV,-5^IV,-6a^IV,-6b^IV,CH₂Ph),4.84(d,1H,J_{A, B}11.2Hz, A of the AB quartet), 4.86(d, J)_1,21.8Hz;H-1^I),4.90(dd,1H;J_1,21.8,J_2,33.1Hz;H-2^III),5.03(d,1H,J_1,21.5Hz;H-1^IV),5.22(dd,1H,J_1,22.1,J_2,32.6Hz;H-2^II),5.27-5.29(m,2H;H-1^III,-1^IV),5.46(dd,1H,J_3,49.7,J_4,59.9Hz;H-4^IV),5.79(dd,1H,J_2,32.9Hz;H-2^IV),5.90-5.96(m,2H;H-4^II,-4^III),7.01-7.56,7.68-8.16(2m,65H;Ar)。

Benzyl 2-O- [ (2,3,4, 6-tetra-O-acetyl- α -D-mannopyranosyl) - (1 → 3) - (2,4, 6-tri-O-benzoyl- α -D-mannopyranosyl) ] - (1 → 3) - (2,4, 6-tri-O-benzoyl- α -D-mannopyranosyl) ] -3,4, 6-tri-O-benzyl- α -D-mannopyranoside (30)

2,3,4, 6-tetra-O-acetyl- α -D-mannopyranosyl trichloroiminoacetate [28 ]](39mg, 78. mu. mol) of a mixture of the alcohol (29) (85mg, 39. mu. mol) in 1,2-DCE (3mL) over molecular sieves (100mg ofPowder) was stirred under argon atmosphere (30 minutes). The mixture was cooled (0 °) with continued stirring (10 min) before addition of TMSOTf (14.2 μ L, 78 μmol). After a period of time (30 min), Et was introduced₃N (100 μ L) and the mixture was filtered. The solvent was evaporated and the residue was treated with FC (30-60% EtOAc/hexanes) to give the tetraacetate (30) as a colorless oil (85mg, 87%).¹H NMR(CDCl₃)1.82-2.04(4s,3H each; CH)₃CO),3.67-3.95,4.05-4.72,4.82-5.03,5.21-5.28,5.69-5.50(m,43H;H-1^I-IV,-2^I-IV,-3^I-IV,4^I-IV,-5^I-IV,-6ab^I-IV,CH₂Ph)，7.01-7.56,7.68-8.16(2m,65H;Ar)。

General procedure for deprotection of the mannooligosaccharides (25,27,29,30)

(A) A small piece of sodium was added to the tetrabenzyl ether (25,27,29,30) solution in MeOH and THF and the combined mixture was stirred (rt, o/n). Thereafter, the mixture was neutralized with Dowex50X8 resin (H +) and filtered. The solvent was evaporated and co-evaporated (MeOH) and used in the following reaction without further purification.

(B) Pd (OH)₂(10% on C) the crude product from (A) was added in THF with H containing a small amount of AcOH (50 □ L)₂O in solution and the combined mixture was stirred vigorously under hydrogen (100p.s.i., 3 hours). Thereafter, the mixture was filtered and the solvent was evaporated. The residue was subjected to gel filtration chromatography (Biogel P2; H)₂O; 60 ml/hr) to give the mannooligosaccharide (8-11) as a colorless powder after freeze-drying. Compounds 8-11 are in all respects consistent with those isolated from the Pichia hydrolysis process (hydolysis) as described above.

Example 2 benzyl glycoside polysulfate (PG500)

Peracetic acid ester 12

The pentasaccharide 11(1.03g, 95% M5), sodium acetate (1.2g) and acetic anhydride (50mL) were heated under stirring at 140 ℃ under a drying tube overnight the mixture was cooled to room temperature, evaporated to dryness, taken up in EtOAc, washed with brine (× 3) and treated with flash chromatography (40g silica gel, 80:20EtOAc: Hx) to give 810mg of the peracetate 12 as a glass with very little pure material.¹H NMR(400MHz,CDCl₃)6.14(d,0.84H,J=2.0,αHl^I),5.71(d,0.16H,J=0.9,βHl^I) 5.30-5.10(m,8H),5.00-4.85(m,7H),4.25-3.70(m,19H),2.20-1.90(m, 51H). For C₆₄H₈₇O₄₃HRMS calculated value of [ M + H ]]⁺1543.4623, found 1543.4599.

General procedure for direct saccharification of fully acetylated oligosaccharides:

the alcohol (6 equivalents) is added to the peracetate (e.g., 12) (1 equivalent) inMS dried DCM solution (0.03M). In some cases, small amounts are addedMS powder. Boron trifluoride etherate (4 equivalents) was added and the mixture was stirred under argon atmosphere at 60 ℃ or 75 ℃ for 2 to 26 hours. The mixture was cooled and triethylamine was added. The mixture was diluted with dichloromethane, washed with saturated aqueous sodium carbonate solution and dried (anhydrous MgSO)₄). The dried solution was filtered and the filter cake was washed with dichloromethane. The combined filtrate and washings were concentrated, loaded onto silica gel and purified by flash chromatography (silica gel, gradient elution with hexane-EtOAc 6:1 to 1: 4) to afford the desired glycoside after evaporation and drying under high vacuum.

Benzyl glycoside 13

The saccharification was performed using 12 and benzyl alcohol to give the product (13) as a colorless colloid, 108mg, 46% (Rf =0.32, hexane-EtOAc =1: 3).¹H NMR(CDCl₃,400MHz)7.35-7.27(m,5H,C₆H₅) 5.30-5.12(m,8H),5.00-4.85(m,8H),4.68(AB quartet, 1H, J =11.8) and 4.50(AB quartet, 1H, J =11.8, PhCH₂O),4.27-3.74(m,19H),2.14(4),2.13(5),2.13,2.10,2.08(4),2.07(9),2.07(6),2.06(9),2.06(6),2.06(2×),2.02,2.00,1.99,1.97,1.94(15s,48H,16×Ac)；¹³C NMR(CDCl₃,100MHz)171.0,170.5(3),170.5(1),170.5(0),170.4,170.3,170.2,170.0(4),170.0(2),169.8(9),169.8(8),169.7,169.6,169.5(6),169.4(6) and 169.3(16 × CO in total), 136.1 (ipso-C)₆H₅) 128.5,128.2 and 127.9(o, m, p-C)₆H₅) 99.2(2C),98.9,98.8,97.3(5 × saccharide-Cl), 76.7,75.1,74.9(9),74.9(7),71.1,70.9,70.8,70.2,69.7,69.5(9),69.5(6),69.4(2),69.3(7),69.2,68.6,68.3,67.1,66.7(3),66.6(7),66.1,65.5,62.4,62.1,61.9,61.6 and 60.2(26C,25 × saccharide carbon excluding 5 × saccharide-Cl and benzyl CH₂) 20.9,20.8(2),20.8(0),20.7(8),20.7,20.6,20.5(4),20.5(1),20.4(9) and 20.4(6) (10C,16 × Ac).

Benzyl glucoside polybasic sulfate (PG500)

Deacetylation of Compound 13 (for polyol C)₃₇H₅₉O₂₆HRMS calculated value of [ M + H ]]⁺919.3296, found 919.3279) and sulphonated according to the general method to give the product (PG500) as a white powder, 76.1mg, 44%.¹H NMR(D₂O,400MHz)7.35-7.26(m,5H,C₆H₅),5.32(s,1H),5.30(d,1H,J=1.2),5.26(d,1H,J=2.0),5.24(d,1H,J=1.6),5.05(dd,1H,J=2.8,2.0),5.00(d,1H,J=2.0),4.87-4.85(m,2H),4.68-4.34(m,12H),4.32-3.86(m,17H)；¹³C NMR(D₂O,100MHz)137.0,129.5,129.4,129.1,100.5(9),100.5(6),100.2,97.9,93.8,76.9,76.8,75.6,75.5(3),75.4(8),74.4,73.8,73.1,73.0,72.8,72.7,71.8,71.3,70.7,70.6,70.4,69.9,69.8,69.7,68.0,67.8,67.5,66.6,66.3(7),66.3(5)。

Example 3 octyl glycoside Polysulfate (PG501)

Octyl glucoside 14

Using 12 and octanolThe saccharification was performed to give the product (14) as a colorless colloid, 207mg, 66% (Rf =0.41, hexane-EtOAc =1: 3).¹H NMR(CDC1₃,400MHz)5.23-5.09(m,8H),4.96-4.82(m,8H),4.23-3.71(m,19H),3.59(dt,1H,J=9.4,6.8,OCH₂R),3.35(dt,1H,J=9.4,6.8,OCH₂R),2.11,2.10(2),2.09(8),2.06,2.05,2.04(4),2.04(1),2.03(8),2.03,2.02,2.01,1.99(3),1.98(8),1.96,1.94 and 1.90(16s,48H,16 × Ac),1.52 (quintuple peak, 2H, J =7.2, CH = 7.2)₂),1.27-1.18(m,10H,(CH₂)₅),0.80(t,3H,J=7.2,CH₃)；¹³C NMR(CDCl₃100MHz)170.4(0) (2C),170.3(8) (2C),170.3,170.2,170.1,169.9(2C),169.8(2),169.7(5),169.6,169.5,169.4(4),169.3(5),169.3(16 × CO,3 overlapped), 99.1(2C),98.8,98.7,98.0(5 × saccharide-Cl), 77.0,75.0,74.8(3),74.7(5),71.0,70.8,70.7,70.1,69.4(9),69.4(7),69.3(0),69.2(7),69.2,68.3,68.2(0),68.1(6),67.2,66.6(4),66.6(0),66.1,65.4, 62.3,61.8 and 61.5(25C, saccharide-C, and octyl saccharide-Cl were excluded₂O),31.5,29.1,29.0,28.9,25.9,22.4(6 × octyl-CH)₂) 20.7(3),20.7(0),20.6(7),20.6,20.5,20.4(3),20.4(0),20.3(9),20.3(7) (9C,16 × Ac),13.85 (octyl-CH)₃)。

Deacetylation of Compound 14 (for polyol C)₃₈H₆₉O₂₆HRMS calculated value of [ M + H ]]⁺941.40784, found 941.4060.) and sulfonated according to the general method to give the product (PG501) as a white powder, 195mg, 72%.¹H NMR(D₂O,400MHz)5.33(s,1H),5.29(d,1H,J=1.6),5.24(d,1H,J=1.6),5.21(d,1H,J=1.6),5.03(dd,1H,J=2.8,2.0),4.87(d,1H,J=1.6),4.86-4.83(m,2H),4.70-3.92(m,27H),3.59(dt,1H,J=9.6,7.0),3.44(dt,1H,J=9.6,7.0),1.48-1.40(m,2H),1.21-1.08(m,10H),0.678(t,3H,J=7.2)；¹³C NMR(D₂O,100MHz)100.5,100.4,100.1,100.0,99.0,98.4(1),98.3(8),98.3(6),98.3(5),76.8(5),76.7(9),76.7,76.6,76.5(2),76.4(7),76.0,75.4(0),75.3(5),75.3,75.2,74.3,73.0(5),72.9(9),72.7,72.6,71.7,70.4,70.2,69.8(4),69.7(5),69.6,69.1,67.8(5),67.7(7),66.5,66.2,31.5,30.0,28.8,25.8,22.5,14.0。

Example 4 PEG₅₀₀₀Polybasic sulfuric acid ester (PG504)

Imidate 15

(A) The acetate (12) (68mg, 51. mu. mol) was reacted with BnNH over a period of time (2 days)₂A mixture of (17. mu.L, 152. mu. mol) in THF (2mL) was stirred (room temperature). The mixture was taken up in CHCl₃The organic phase was evaporated and co-evaporated (2 × 10mL MeCN) and used in the next reaction without further purification.

(B) DBU (10 μ L, 6.7 μmol) was added to a solution of the crude product (from a) and trichloroacetonitrile (1.0mL, 10mmol) in 1,2-DCE (4mL) and the combined mixture was stirred (0 ℃ → 12 ℃, o/n). The mixture was concentrated and the residue was subjected to FC (50-90% EtOAc/hexanes) to give 15 as a pale yellow oil (35mg, 48%, 2 steps).¹H NMR(400MHz,CDCl₃)8.70(s,1H,NH),6.32(d,1H,J=2.0,H1^I),5.36-5.13(m,8H),5.00-4.90(m,6H),4.26-3.75(m,20H),2.15-1.94(m,48H)。

PEG₅₀₀₀Polybasic sulfuric acid ester (PG504)

(A) The imidate 15(33mg, 20.2. mu. mol) was reacted with PEG₅₀₀₀Mixture of monomethyl ether (151mg, 30.3. mu. mol) in 1,2-DCE (3mL) over molecular sieves (50mg ofPowder) was stirred under argon atmosphere (10 minutes). The mixture was cooled (-20 ℃) with continued stirring (10 min) before TMSOTf (5. mu.L, 2.8. mu. mol) was added. After a period of time (20 min), Et was introduced₃N (10 μ L) and the mixture was filtered. The solvent was evaporated and the residue was subjected to FC (0-7.5% MeOH/CHCl)₃) Treatment gave 16(104mg,80%, based on average M) as a colorless glass_r6483)。¹HNMR(400MHz,CDCl₃)5.28-4.87(m,14H),4.43-3.42(m,829H,),3.34(s,3H,OMe),2.15-1.94(m,48H)。

(B) Compound 16(104mg, 16. mu. mol) was deacetylated according to the general procedure to give Man as a colorless wax₅-PEG₅₀₀₀OMe (82mg, 89%, based on the mean M)_r5769)。

(C) Subjecting said M to a general procedure₅-PEG₅₀₀₀OMe (82mg, 14. mu. mol) to give PG504 as a colorless foam (45mg, 42% based on average M)_r7401)。¹H NMR(400MHz,D₂O)5.34-4.87(m,7H),4.71-3.97(m,20H),3.76-3.35(m,432H),3.23(s,3H,OMe)。

Example 5: PEG₂₀₀₀Polybasic sulfuric acid ester (PG506)

(A) Such as PEG₅₀₀₀Treatment of the imidate (15) (60mg, 36.5. mu. mol) with PEG as described by OMe with TMSOTf₂₀₀₀A mixture of OMe (110mg, 55.0 μmol) to give compound 17(96mg, 74%) as a colorless glass.¹HNMR(400MHz,CDCl₃)5.28-5.13,5.00-4.87,4.27-3.40(3m, many H, H1)^I-V,2^I-V,3^I-V,4^I-V,5^{I -V},6a^I-V,6b^I-V,OCH₂CH₂O),3.34(s,3H, OMe),2.15-1.94(16s,3H per unitOne, COMe).

(B) Compound 17 is deacetylated according to the general procedure to give colorless waxy PEG₂₀₀₀OMe polyol (63mg, 81%). The residue was used in the next reaction without further purification or characterization.

(C) The product from (B) above was sulfonated according to a general method to give the title compound (PG506) (47mg, 68%) as a colorless powder.¹H NMR(400MHz,D₂O)5.34-3.97(m,498H),3.80-3.35(m,81H),3.23(s,3H,OMe)。

Example 6: PG502

Azide 19

Peracetate 12(270mg, 175. mu. mol), TMSN₃(60mg, 525. mu. mol) and SnCl₄A solution of (200. mu.L of 1M in DCM) in dry DCM (20mL) was stirred overnight in the dark. An additional amount (3 equivalents) of TMSN was added₃And SnCl₄And stirring was continued overnight again in the dark. Ice and NaHCO were added₃(saturated aqueous solution) and the mixture was extracted with EtOAc, washed with brine, evaporated and treated with flash chromatography (10g silica gel, gradient elution, 50:50 to 75:25EtOAc: Hx) to give 218mg (82%) of azide 19.¹H NMR(400MHz,CDCl₃)5.52(d,1H,J=2.0,Hl^I),5.29-5.12(m,8H),5.02-4.87(m,7H),4.29-3.76(m,19H),2.18-1.95(m,48H)；¹³C NMR(100MHz,CDCl₃)170.5(9),170.5(7),170.5(6),170.4,170.3,170.2,170.1,169.9(9),169.9(8),169.9(5),169.7(3),169.6(9),169.6(6),169.6,169.5,169.3,99.3(0),99.2(7),99.1,99.0,88.1,75.2,75.1,74.8,71.1,70.9,70.8,70.6,69.7,69.5,69.4,69.2,68.3,67.3,66.8,66.7,65.5(9),65.5(8),62.6,62.2,62.0,61.7,20.8(8),20.8(6),20.8,20.7,20.6(2),20.5(8),20.5(7),20.5. For C₆₂H₈₄N₃O₄₁HRMS calculated value of [ M + H ]]⁺1526.4583, found 1526.4557.

1-deoxy-1-alpha-phenoxyacetamido peracetate 21

19(32mg, 21. mu. mol), PPh₃A solution of (11mg, 42.6. mu. mol) and phenoxyacetyl chloride (7.3mg, 43. mu. mol) in anhydrous acetonitrile (5mL) was stirred at 0 ℃ for 4 hours and then at room temperature overnight. Addition of EtOAc and NaHCO₃(saturated aqueous solution) and the organic layer was washed with brine and then dried (MgSO₄) And flash chromatography (gradient elution 60:40 to 90:10EtOAc: Hx) afforded 11.4mg (33%) of the final product with some residual PPh₃/PPh₃Amide 21 of O.¹H NMR(400MHz,CDCl₃)7.36-7.32(m,2H),7.18(br d,1H,J=8.1,NH),7.00-6.90(m,3H),5.79(dd,1H,J=3.8,8.2,H1^I) 5.32-4.97(m,15H),4.60-3.76(m,21H),2.20-1.95(m,48H, AcO). For C₇₀H₉₂NO₄₃HRMS calculated value of [ M + H ]]⁺1634.5045, found 1634.5002.

PG502

The peracetate 21(11mg, 6.7 μmol) was deacetylated and sulfonated according to the general method to yield 6mg (34% for 2 steps) of PG502 after lyophilization.¹H NMR(400MHz,D₂O, solvent compressed) 7.30-7.21(m,2H, ArH)^m),6.96-6.84(m,3H,ArH^o,p) 5.56-3.59(m,30H affected by compression).

Example 7: PG503

1-deoxy-1-alpha-biotin aminocaproamide peracetate 22

A mixture of 19(70mg, 46. mu. mol) and Adam catalyst (2mg) in 2:1EtOAc: EtOH (3mL) in H₂Stirred overnight (100psi), then filtered, evaporated and co-evaporated with anhydrous pyridine. Biotin aminocaproic acid N-hydroxysuccinimide ester (31mg, 68. mu. mol) and 1mL of anhydrous pyridine were added and the mixture was heated to 60 ℃ for 3 days with stirring. The solution was evaporated and flash chromatographed (9.4 gEt)₃N-washed silica gel, gradient elution 75:25EtOAc: Hx to 30:70MeOH: EtOAc) afforded 30.8mg (36%, over two steps) of amide 22.¹H NMR(400MHz,CDCl₃)7.41(brd,1H, J =9.4, NH),6.47,6.17(2 × br s,2 × 1H, imide NHs),5.40(brd,1H, J =9.4, H1)^I) 5.40-4.90(m,16H),4.52(dd,1H, J =4.9,7.5, biotin-H4), 4.36-3.72(m,20H),3.25-3.12(m,3H),2.91(dd,1H, J =5.0,13.0, biotin-H5A), 2.75(d,1H, J =12.9, biotin-H5B), 2.27-1.96(m,52H),1.82-1.29(m,12H, alkyl chain).

PG503

The peracetate 22(30mg, 16.3. mu. mol) was deacetylated and sulphonated according to the general method to yield 28mg (61% for 2 steps) of PG503 after freeze-drying.¹H NMR(400MHz,D₂O, solvent compressed, affected by amide rotamers) 5.60-4.75(m,7H, saccharide Hs),4.68(dd,1H, J =4.7,7.2, biotin-H4), 4.60-3.60(m,26H, saccharide Hs),4.21(dd,1H, J =4.4,7.2, biotin-H3), 3.33-3.16(m,1H, biotin-H2), 3.07-2.97(m,3H, biotin-H5A + CH 3), 3.07-2.97(m,3H, biotin-H A + CH 3526)₂N),2.92(dd,1H, J =4.9,13.5, biotin-H5B), 2.33-2.14(m,2H, COCH)₂B),2.09(t,2H,J=7.4,COCH₂A) 1.63-1.15(m,12H, alkyl chain).

Example 8: PG505

An azide 31.

Mixing maltose hexaose peracetate (500mg, 273. mu. mol) and TMSN₃(83mg, 726. mu. mol) and SnCl₄A solution of (1M in DCM, 145. mu.L) in dry DCM (20mL) was stirred overnight in the dark. Adding additional amount of TMSN₃(50. mu.L) and SnCl₄(1M in DCM, 100. mu.L) and stirring again overnight in the dark is continued. Ice and NaHCO were added₃(saturated aqueous solution) and the mixture was extracted with EtOAc, washed with brine, evaporated and treated with flash chromatography (10g silica gel, gradient elution, 75:20 to 80:20EtOAc: Hx) to give 488mg (98%) of azide 31.¹H NMR(400MHz,CDCl₃):5.30-5.11(m,11H),4.93(t,1H,J=9.9),4.72(dd,1H,J=4.0,10.5),4.68-4.57(m,6H),4.44-3.67(m,23H),2.09-1.85(m,57H)。¹³C NMR(100MHz,CDCl₃):170.3(4),170.3(1),170.2(7),170.2,170.1(4),170.1(0),170.0(7),170.0,169.6,169.4,169.3,169.2(3),169.2(2),169.1(7),169.1(4),169.1(1),95.5(0),95.4(5),95.4,95.3,87.1,74.7,73.9,73.3,73.2,72.2,71.4,71.3,71.2(4),71.2(1),70.2,70.1,69.8,69.0,68.8,68.7,68.2,67.7,62.4,62.3,62.1(8),62.1(6),62.0,61.1,30.0,20.5(5),20.5(3),20.5(0),20.4(6),20.3(3),20.2(8),20.2(4),20.2(2)。

PG505

The azide 31(97mg,54 μmol) was deacetylated and sulphonated according to the general method to give 66mg (41% for 2 steps) of PG505 after freeze drying.¹H NMR(400MHz,D₂O, dissolvingCompressed) 3.69-5.78(m,42H affected by solvent compression).

Example 9: PG515

6-Azide-6-deoxy-2, 3, 4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroiminoacetate (34)

(A) H is to be₂SO₄(0.5mL) was added to the methyl glycoside (32) [29 ]](1.52g, 2.9mmol) with Ac₂A cooled (0 °) solution of O (10mL) in AcOH (5mL) and the combined mixture was stirred (0 ° → room temperature, O/n). NaOAc (1.0g) was added in portions until pH>5.0 and then treating the mixture with MeOH (3 mL). The mixture was filtered and the solvent was evaporated and co-evaporated (toluene) before workup (EtOAc) and RSF (10-20% EtOAc/hexanes) to give the acetate (33) (1.12g, 70%) as a colorless foam.

(B) Hydrazine acetate (196mg, 2.13mmol) was added to a stirred solution of the acetate (33) (1.08g, 1.94mmol) in DMF (10mL) and the combined mixture was heated (55 °,15 min.) the mixture was poured onto saturated NaCl and extracted (EtOAc.) the organic layer was evaporated and treated with RSF (10-30% EtOAc/hexanes) to give a colorless oil (888 mg.) the residue was co-evaporated (2 × 100mL CH 100 mL)₃CN) and used in the next reaction without further purification or performance description.

(C) DBU (3 drops) was added to the crude product (888mg) and Cl from (B) (above)₃CN (2.0mL, 20mmol) in 1,2-DCE (8mL) and the combined mixture was stirred (0 ° → room temperature, 1 h). The mixture was filtered, the solvent was evaporated and the residue was treated with FC (10-30% EtOAc/hexanes) to give the imidate (34) as a colorless oil (777mg, 61%, 2 steps).¹H NMR(400MHz,CDCl₃)8.88(br s,1H,NH),8.10-7.22(m,15H,ArH),6.56(d,1H,J_1,22.0Hz,H1),5.99(dd,1H,J_3,4-4,59.6Hz,H4),5.94-5.88(m,2H,H2,3),4.44(ddd,1H,J_5,62.8,5.6Hz,H5),3.54(dd,1H,J_6,613.6Hz,H6),3.47(dd,1H,H6)。¹³C NMR(100MHz,CDCl₃)165.61,165.37,159.95,134.00,133.92,133.58,130.25,130.05,129.12,129.04,128.97,128.91,128.76,128.74,128.57,94.62,73.03,69.69,68.90,67.05,51.06。

Benzyl (6-azido-6-deoxy- α -D-mannopyranosyl) - (1 → 3) - (α -D-mannopyranosyl) - (1 → 2) - (α -D-mannopyranoside) (37)

(A) Mixing the imidate (34) (93mg, 141. mu. mol), the alcohol (35) (90mg, 94.1. mu. mol) and molecular sieves (50mg ofPowder) mixture in 1,2-DCE (3mL) was treated with TMSOTf (10 μ L, 55.1 μmol) and the combined mixture was stirred (0 ° → room temperature, 20 min). Introduction of Et₃N (100 μ L), filtering the mixture and evaporating the solvent. The residue was FC (10-40% EtOAc/hexanes) treated to give the azide (36) as a colorless oil (68mg, 57%).¹H NMR(400MHz,CDCl₃)8.80-7.12(m,65H,ArH),6.01(dd,1H,J_3,4-4,59.9Hz,H4^III),5.96(dd,1H,J_3,4-4,59.9Hz,H4^I),5.92(dd,1H,J_3,4-4,59.6Hz,H4^II),5.83(dd,1H,J_{2, 3}3.3Hz,H3^I),5.79(dd,1H,J_l,22.0,J_2,33.3Hz,H2^II),5.70(dd,1H,J_3,4-4,59.9Hz,H4^IV),5.50(dd,1H,J_2,33.3Hz,H3^IV),5.36(d,1H,J_1,21.7Hz,Hl^III),5.29(dd,1H,J_2,33.0Hz,H2^III),5.23(d,1H,H1^II),5.18(dd,1H,J_1,21.9Hz,H2^IV),5.16(d,1H,J_1,21.6Hz,H1^I),4.87(d,1H,H1^IV),4.72-4.24(m,14H,H2^I,H3^II,III,H5^I-III,H6^I-III),3.99(ddd,1H,J_5,62.9,3.4Hz,H5^IV),3.02(dd,1H,J_6,613.5Hz,H6^IV),2,83(dd,1H,H6^IV)。

(B) The benzoate ester (36) (63mg, 31. mu. mol) was transesterified according to the general procedure and the residue was chromatographed (C18, 0-10% MeOH/H)₂O) to give the tetrasaccharide (37) as a colorless glass (15mg, 62%).¹H NMR(400MHz,MeOD)7.34-7.22(m,5H,ArH),5.12(d,1H,J_l,21.5Hz,Hla),5.09(d,1H,J_1,21.7Hz,Hlb),5.07(d,1H,J_1,21.6Hz,H1c),4.92(d,1H,J_1,21.9Hz, Hld),4.71,4.48(AB of the AB quartet, J11.7Hz, CH₂Ph),4.14(dd,1H,J_2,33.0Hz,H2a),4.19(dd,1H,J_2,33.2Hz,H2b),3.96(dd,1H,J_2,33.4Hz,H2c),3.94(dd,1H,J_3,49.4Hz,H3b),3.88-3.52(m,19H,H2d,H3a,c,d,H4a-d,H5a-d,H6a-d),3.44(dd,1H,J_5,66.3,J_6,610.1Hz,H6^IV)。

PG515

The tetrasaccharide 37(12mg, 15.3. mu. mol) was sulfonated according to the general method to give 14mg (38% for 2 steps) of PG515 after freeze-drying.¹H NMR(500MHz,D₂O)7.47-7.37(m,1H,ArH),5.45-4.02(m,29H,C1^I-IV,2^I-IV,3^I-IV,4^I-IV,5^I-IV,6a^I-IV,6b^1-III,CH₂Ph),3.69-3.67(m,1H,H6b^IV)。

Example 10: PG509

Methyl 3-O- (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) -2,4, 6-tri-O-benzoyl-alpha-D-mannopyranoside (39)

(A) 3-O-allyl-2, 4, 6-tri-O-benzoyl- α -D-mannopyranosyl trichloroacetimidate [26 ]](410mg,0.57mmol) with methyl 2,4, 6-tri-O-benzoyl- α -D-mannopyranoside [26 ]](300mg,0.51mmol) of a mixture in 1,2-DCE (6mL) over molecular sieves (700mg ofPowder) was treated with TMSOTf (30 μ L, 0.17mmol) and the combined mixture was stirred (0 ° → room temperature, 30 min). Introduction of Et₃N (100 μ L), filtering the mixture and evaporating the solvent. The residue was subjected to FC (10-50% EtOAc/hexanes) to give disaccharide 38 as a colorless oil.

(B) PdCl₂(40mg) was added to a solution of the product from (A) in MeOH (10mL) and 1,2-DCE (10mL) and the combined mixture was heated (70, 40 min). The solvent was evaporated and the residue was subjected to FC (10-50% EtOAc/hexanes) to give the alcohol (39) as a colorless oil (316mg, 68%, 2 steps). The above-mentioned¹H and¹³CNMR(CDCl₃) Spectrum and in document [26 ]]Similar to those already reported in (a).

Methyl (alpha-D-mannopyranosyl) - (1 → 3) - (alpha-D-mannopyranoside) (40)

The alcohol (39) (10mg, 0.10mmol) was transesterified according to the general method to give the disaccharide (40) (3mg, 85%) as a colorless oil, which was confirmed by NMR reported in the literature [30, 31 ].

PG509.

The disaccharide 40(25mg, 70 μmol) was sulfonated according to the general procedure to give 27mg (36%) of PG509 after freeze-drying.¹H NMR(400MHz,D₂O)5.26(d,1H,J_1,21.8Hz;Hl^II),4.98(dd,1H,J_2,32.4Hz;H2^II),4.87(d,1H,J_1,21.9Hz;Hl^I),4.60-4.55(m,1H;H3^II),4.53(dd,1H,J_2,32.3Hz;H2^I),4.41-4.19(m,5H;H4^I,4^II,6a^I,6a^II,6b^II),4.15(dd,1H,J_3,49.3Hz;H3^I),4.06-3.91(m,3H;H5^I,5^II,6b^I),3.29(s,3H;OCH₃)。

Example 11: PG508

Methyl 3-O- [3-O- (2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl) -2,4, 6-tri-O-benzoyl-alpha-D-mannopyranosyl ] -2,4, 6-tri-O-benzoyl-alpha-D-mannopyranoside (42)

(A) A mixture of 3-O-allyl-2, 4, 6-tri-O-benzoyl- α -D-mannopyranosyl trichloroacetimidate (269mg, 0.37mmol) and the alcohol (39) (306mg, 0.31mmol) in 1,2-DCE (5mL) was placed over molecular sieves (100mg ofPowder) was treated with TMSOTf (20 μ L, 0.11mmol) and the combined mixture was stirred (0 ° → room temperature, 30 min). Introduction of Et₃N (100 μ L), filtering the mixture and evaporating the solvent. The residue was subjected to FC (10-50% EtOAc/hexanes) treatmentThereby presumably obtaining the trisaccharide 41 as a colorless oil.

(B) PdCl₂(40mg) was added to a solution of the product from (A) in MeOH (10mL) and 1,2-DCE (10mL) and the mixture was heated (70, 40 min). The solvent was evaporated and the residue was subjected to FC (10-50% EtOAc/hexanes) to give the alcohol (42) as a colorless oil (316g, 70%, 2 steps).¹H NMR(400MHz,CDCl₃)8.14-7.22(m,45H,ArH),6.63(dd,1H,J_1III,2III1.8,J_2III,3III3.3Hz,H2^III),5.94(dd,1H,J_3III,4III10.0,J_4III,5III10.0Hz,H4^III),5.84(dd,1H,J_3II,4II9.9,J_4II,5II9.9Hz,H4^II),5.48(dd,1H,J_3I,4I9.8,J_4I,5I9.8Hz,H4^I),5.26(d,1H,J_1I,2I1.9Hz,H1^I),5.22(dd,1H,J_1II,2II2.1,J_2II,3II3.0Hz,H2^II),4.91(d,1H,H1^III),4.90(dd,1H,J_2I,3I3.2Hz,H2^I),4.86(dd,1H,J_{1II, 2II}1.7Hz,H1^II),4.67-4.63(,12H,H3^I,3^II,3^III,5^I,5^II,5^III,6^I,6^II,6^III)。¹³CNMR(100MHz,CDCl₃)166.49,166.38,166.25,166.07,165.94,165.77,165.63,165.19,165.15,133.80,133.60,133.61,133.58,133.52,133.06,130.22,130.16,130.09,130.05,130.16,129.97,129.9,129.88,129.84,129.51,129.17,129.01,128.85,128.63,128.53,128.5,128.46,99.35,99.24,98.73,76.48,76.12,72.45,71.77,71.64,69.93,69.7,69.01,68.86,68.6,68.53,67.82,63.17,62.79,62.41,55.66；ESMS:m/z1373.4[M-Bz+H+Na]⁺，1269.4[M-2Bz+2H+Na]⁺。

Methyl (α -D-mannopyranosyl) - (1 → 3) - (α -D-mannopyranoside) (43)

The alcohol (42) (115mg,0.79mmol) was transesterified according to the general method to give the trisaccharide (43) (35mg,86%) as a colorless oil according to the literature [32 ]]NMR confirmation reported in (1). HRMS M/z519.1862[ M + H ]]⁺,541.1646[M+Na]⁺。

PG508.

The trisaccharide 43(25mg, 49. mu. mol) was sulfonated according to the general method to give 36mg (49%) of PG508 after freeze-drying.¹H NMR(400MHz,D₂O)5.26(d,1H,J_1,21.9Hz;H1^III),5.22(d,1H,J_1,21.8Hz;H1^II),5.04(dd,1H,J_2,32.4Hz;H2^III),4.89(d,1H,J_1,21.6Hz;H1^I),4.76-4.75(m,1H;H2^II),4.60-4.55(m,1H;H3^III),4.55(dd,1H,J_2,33.1Hz;H2^I),4.50(dd,1H,J_3,49.6,J_4,59.7Hz;H4^III),4.41-4.12,4.04-3.91(m,12H;H3^II,4^I,4^II,5^I-III,6a^I-III,6b^I-III),4.10(dd,1H,J_{3, 4}9.5Hz;H3^I),3.29(s,3H;OCH₃)。

Example 12: PG512

Benzyl (3-O-allyl- α -D-mannopyranosyl) - (1 → 3) - (α -D-mannopyranosyl) - (l → 2) - (3,4, 6-tri-O-benzyl- α -D-mannopyranoside) (44)

Sodium (small pieces) was added to the nonabenzoate (28) (115mg,0.79mmol) in MeOH (6mL) and the combined mixture was stirred (room temperature, o/n).The mixture was neutralized (Dowex50X8, H)⁺) Filtered and the filtrate concentrated and subjected to FC (0-10% MeOH/CH)₂Cl₂) Work-up gave the tetrabenzyl ether (44) (89mg, 64%) as a colourless oil.¹H NMR(CD₃OD)7.33-7.13(m,20H,ArH),6.02-5.92(m,1H,CH=CH₂),5.32-5.27,5.11-5.09(2m,2H,CH=CH₂),5.10(d,1H,J_1,21.4Hz,Hla),5.09(d,1H,J_1,21.5Hz,Hlb),5.03(d,1H,J_1,21.2Hz,H1c),4.97(d,1H,J_1,21.4Hz, Hld),4.74,4.49(2d, AB, J of ABq)_{H, H}10.9Hz,PhCH₂A),4.67,4.48(2d, AB, J of ABq)_H,H11.8Hz,PhCH₂B),4.65,4.58(2d, AB, J of ABq)_H,H11.6Hz,PhCH₂C),4.57,4.51(2d, AB, J of ABq)_H,H12.4Hz,PhCH₂-d),4.21-3.62(m,26H,H2^I-IV,3^I-IV,4^I-IV,5^I-IV,6a^I-IV,6b^I-IV,OCH₂CH=)。

PG512

The tetrasaccharide 44(23mg, 21.5. mu. mol) was sulfonated according to the general method to give PG512(26mg, 61%) as a colorless powder.¹H NMR(400MHz,D₂O)7.32-7.18,7.00-6.98(2m,20H,ArH),5.88-5.78(m,1H,CH=CH₂),5.30-5.23,5.08-5.04,4.91-4.90,4.83-4.82,4.71-4.08,4.00-3.89,3.73-3.70,3.62-3.45(8m,40H,CH=CH₂,OCH₂CH,H1-6^I-IV,PhCH₂ ^I-IV)。

Example 13: PG513

The tetrabenzyl ether (44) (62mg, 50. mu. mol)And Pd (OH)₂(10mg of 10% on C) in THF (1mL) and H₂Mixture of O (1mL) in H₂(100p.s.i.) (room temperature o/n) under stirring. The mixture was filtered, concentrated and subjected to FC (SiO)₂；H₂O) to give the propyl ether (45) as a colorless glass (32mg, 73%).¹H NMR(D₂O)5.22(br s,1H,Hla),5.00(d,1H,J_1,21.7Hz,Hlb),4.97(d,1H,J_1,21.6Hz,H1c),4.87(d,1H,J_{1, 2}1.8Hz,H1d),4.11-4.07,3.91-3.35(2m,26H,H2^I-IV,3^I-IV,4^I-IV,5^I-IV,6a^I-IV,6b^I-IV,OCH₂),1.50-1.42(m,2H,CH₂CH₃),0.76(t,3H,J_H,H7.2Hz,CH₂CH₃)。

PG513

The tetrasaccharide 45(21mg, 29.6. mu. mol) was sulfonated according to the general method to give PG513(29mg, 34%) as a colorless powder.¹H NMR(D₂O)5.61(d,1H,J_1,22.3Hz;H1a),5.61(br s,1H;H1b),5.32(d,1H,J_{1, 2}1.8Hz;H1c),5.26(d,1H,J_1,22.0Hz;H1d),4.90-4.88,4.77-4.31,4.23-4.04,3.98-3.81,3.57-3.51,3.41-3.36(6m,26H,OCH₂CH₂,H2-6^I-IV),1.48-1.39(m,1H;CH₂CH₃),0.76(dd,1H,J_H,H7.4Hz;CH₂CH₃)。

Example 14: PG510

The polyol 46[31 ] is reacted according to the general method](22mg, 61.7. mu. mol) to give PG510(46mg, 7. mu. mol) as a colorless powder0%)。¹H NMR(D₂O)5.10(d,1H,J_1,22.0Hz;H1^II),4.90(d,1H,J_1,22.0Hz;H1^I),4.78(dd,1H,J_2,33.0Hz;H2^II),4.73(dd,1H,J_2,33.1Hz;H2^I),4.64-4.40(m,1H;H3^II),4.52(dd,1H,J_3,49.5Hz;H3^I),4.33-4.30(m,2H;H4^II,6a^II),4.22(dd,1H,J_4,59.7Hz;H4^I),4.12-4.04(m,2H;H5^II,6b^II),3.96-3.90(m,2H;H5^I,6a^I),3.76(dd,1H,J_5,6b8.6,J_6a,6b11.3Hz;H6b^I),3.31(s,3H;OCH₃).

Example 15: PG511

The polyol 47[31 ] is reacted according to the general method](20mg, 56. mu. mol) was sulfonated to give PG511(29mg, 48%) as a colorless powder.¹H NMR(D₂O)5.36(d,1H,J_1,22.2Hz;H1^II),4.90(br s,1H;H2^II),4.87(d,1H,J_1,22.1Hz;H1^I),4.74(dd,1H,J_2,33.0Hz;H2^II),4.58-4.40,4.29-4.10,3.88-3.85(3m,10H,H3-6^I,II),3.30(s,3H;OCH₃)。

Example 16: PG514

Azide 18

(A) Boron trifluoride diethyl etherate (257mg, 1.81mmol) was slowly added to the reaction mixture of the peracetate 12(700mg, 0.453mmol) and 6-bromo-1-hexanol (492.7mg, 2.721mmol) in DCE (20mL,molecular sieve) and the mixture was stirred under argon at 60 ℃ for 72 hours. The solution was cooled and Et₃N neutralized, diluted with DCM (30mL) and saturated NaHCO₃Washed and dried (MgSO)₄) And flash chromatography (silica gel, gradient elution, 40:60 to 100:0EtOAc: Hx) afforded 340mg (0.204mmol, 45.0%) of the 6-bromohexyl glycoside.¹H NMR(400MHz,CDCl₃):5.25-5.08(m,8H),4.98-4.81(m,8H),4.25-3.70(m,19H),3.607(dt,1H,J=9.553,J=6.635,OCH₂A),3.354(dt,1H,J=9.641,J=6.637,OCH₂B),3.33(t,2H,J=6.700,CH₂Br),2.104,2.096,2.09,2.06,2.043,2.038,2.036,2.033,2.029,2.02,2.01,1.97,1.95,1.94 and 1.90(16x S,48H, OAc),1.85-1.74(m,2H, CH, oh)₂),1.59-1.46(m,2H,CH₂),1.44-1.35(m,2H,CH₂),1.35-1.25(m,2H,CH₂)；¹³C NMR(CDCl₃100MHz) 170.42,170.41,170.39,170.28,170.16,170.07,169.96,169.94,169.83,169.77,169.58,169.52,169.45,169.36,169.25(19xCO),99.10,98.83,98.75,98.01 (saccharide-C1), 76.96,75.00,74.83,74.75,70.96,70.82,70.70,70.08,69.49,69.28,69.16,68.24,68.17,68.04,67.20,66.65,66.60,66.09,65.44,62.41,62.31,61.86, and 61.54 (saccharide carbons exclude saccharide-Cl and bromohexyl-CH₂O),33.49,32.32,29.43,28.92,27.59,25.12(6x bromohexyl-CH)₂),20.73,20.71,20.68,20.62,20.56,20.47,20.44,20.41,(Ac-CH₃),13.85(CH₂Br)。

(B) 6-bromohexyl glycoside from (A) (340mg, 0.204mmol) was heated with a solution of sodium azide (66mg,1.02mmol) in DMF (4mL) at 100 ℃ for 48 hours. TLC analysis of the crude mixture showed no change. Tetrabutylammonium iodide (20mg) was then added and the mixture was allowed to react for further 48 hours. The crude mixture was cooled and flash chromatographed (0:100 to 5:95DCM: MeOH) to afford 21.1mg (0.013mmol, 6.4%) of azide 18.

PG514

(A) The azide 18(21.1mg, 0.013mmol) was deacetylated under standard zemplian conditions (2mL MeOH) to afford 12.6mg (0.013mmol, 102%) of polyol 48.

(B) With SO according to the general sulphation method₃Trimethylamine treatment of the polyol 48(12.6mg, 13.2. mu. mol) gave PG514(18.4mg, 54%) as a colorless powder.¹H NMR(D₂O,400MHz):5.40-4.69(m,8H),4.68-3.41(m,27H),3.22(t,2H,J=6.5),1.51(br s,5H),1.29(br s,5H)。

Biological assay for compounds

Growth factor binding assay

The binding affinities of ligands to the growth factors FGF-1, FGF-2 and VEGF were measured using a Surface Plasmon Resonance (SPR) based solution affinity assay. The principle of the test is as follows: heparin immobilized on the sensor chip surface distinguishes between free and bound growth factors in the equilibrium solution of growth factors and ligands. When the solution is injected, the free growth factor binds to the immobilized heparin, detected as an increase in SPR response and therefrom determining its concentration. The decrease in free growth factor concentration as a function of the ligand concentration calculates the dissociation constant, K_d. It is important to note that when the interaction involves the HS binding site, only ligands bound to the growth factor can be detected, thus excluding the possibility of evaluating non-specific binding to other sites on the protein. It has been assumed that the ligand interaction is a 1:1 stoichiometric relationship for all proteins.

For the test of the growth factor binding activity, a heparin-coated sensor chip was used. The preparation of sensor chips coated with streptavidin by immobilization of biotinylated BSA-heparin has been described.[5]Heparin has also been immobilized by aldehyde coupling using adipic acid dihydrazide or 1, 4-diaminobutane. For each K_dFor measurements, solutions were prepared containing a fixed concentration of protein and varying concentrations of the ligand in a buffer. The ligands binding to FGF-1 and VEGF were measured in HBS-EP buffer (10mM HEPES, pH7.4, 150mM NaCl, 3.0mM EDTA and 0.005% (v/v) polysorbate 20), while the ligand binding to FGF-2 was measured in HBS-EP buffer containing 0.3M NaCl. [5]Prior to injection, the samples were maintained at 4 ℃ to maximize protein stability. For each test mixture, 50-200 μ L of solution was injected at 5-40 μ L/min and the relative binding response was measured. All surface binding assays were performed at 25 ℃. The surface was regenerated by injecting 40. mu.L of 4M NaC1 at 40. mu.L/min and then 40. mu.L of buffer at 40. mu.L/min.

Sensorgram (sensorgram) data were analyzed using BIA evaluation software (BIAcore). Background sensor grams were subtracted from experimental sensor grams to obtain the specifically bound curves, and then the baselines of all curves were zeroed. The standard curve associated with the protein concentration of the injection solution versus the relative response value is linear, indicating that the binding response is directly proportional to the protein concentration, and thus that the binding assay was performed under mass transfer conditions. [34] Thus, the equation can be used to convert the relative binding response of each injection to the concentration of free protein.

Wherein r is the relative binding response and r_mIs the maximum binding response.

The equilibrium of binding established in the solution prior to injection was assumed to be a 1:1 stoichiometric relationship. Therefore, for the said balance,

wherein P corresponds to said growth factor protein, L is said ligand, and P.L is said protein-ligand complex, said equilibrium equation is

And the binding equation [5] can be expressed as

Given K_dThe value corresponding to [ P ] when using said binding equation]To [ L ]]_{Total of}The value of the region of (a). Wherein K_dValues are measured in duplicate, which represent the average of the repeated measurements. GAG mimetics, e.g., PI-88, that bind tightly to these growth factors have been shown to produce biological effects in vivo. [5]Heparanase inhibition assay

The heparanase assay was performed using the Microcon ultrafiltration assay. The assay relies on the principle of physically isolated Heparan Sulfate (HS) that has been digested by heparanase from native HS to determine heparanase activity. The assay used an ultrafiltration device (Microcon YM-10) to isolate smaller heparanase-broken HS fragments from native HS.

The reaction was set up in a volume of 90 μ L,

40mM acetate buffer (pH5.0)

0.1mg/mL BSA

90ng heparanase

2.5μM³H-labelled HS

Various concentrations of inhibitor.

Establishing the reaction with all components except³H-labeled HS, and allowed to equilibrate at 22 ℃ for 10 minutes. The assay was then started by adding the HS and immediately 20. mu.L was removed, mixed with 80. mu.L of 10mM phosphate (pH7.0) and the 100. mu.L was transferred into a Microcon YM-10 concentrator, which was then centrifuged at about 14000g for 5 minutes. The solution (filtrate) that passed through the membrane was retained. The sample was designated as the time =0 sample. The assay (now 70 μ L in volume) was allowed to react at 22 ℃ for 2.5 hours and then the filtration step was repeated for three 20 μ L aliquots of each assay.

Calculating the time =0 filtrate and the three 2.5 hour filtrate samples³H. The difference between the time =0 and the average 2.5 hour sample gives the amount of heparanase activity. All inhibition assays were run as heparanase standard assays, consistent with the test components above except that no inhibitor was present and the amount of heparanase inhibition in the other assay was determined by comparison to the standard. IC of PI-88 in this experiment₅₀Is 0.98. mu.M.

Antiviral assay

Consistently using monolayer cultures of vero cells [35 [ ]]With herpes simplex virus (HSV-1) KOS321 strain [36 ]]. Antiviral assays for the compounds were performed as described by Nyberg et al. [13]Briefly, the effect of compounds on the infection of cells by exogenously added viruses was tested by mixing five-fold serial dilutions of the compounds (at 0.032-20 μ M) with approximately 200 plaque forming units of the virus. The virus and compound were then incubated at room temperature for 10 minutes, the mixture was added to the cells and left on the cell monolayer for 2 hours at 37 ℃. Next, the inoculum was withdrawn and used in an overlapping culture of a 1% methylcellulose solution in Eagle's Minimal Essential Medium (EMEM)And (5) replacing nutrient. The viral plaques generated after 3 days of cell culture at 37 ℃ were stained with a 1% crystal violet solution and counted. The effect of the compounds on the cell-to-cell spread of HSV-1 was tested by adding to the cells a five-fold dilution series of the compounds (at 0.032-20 μ M) in the serum-free overlay medium after infecting them with HSV-1. After incubation of the compound with the cells at 37 ℃ for 3 days, images of 20 plaques were obtained and area measurements were performed using IM500 software (Leica). The results of viral infection of cells and cell-to-cell transmission of viruses are shown in FIGS. 1A and 1B, respectively, and the derived IC' s₅₀The values are listed in table 1.

Results

The results of the tests as described in the previous section are listed in table 1.

TABLE 1

Pharmacokinetic evaluation

Preparation of³⁵S]-labelled compounds

Under vacuum with P₂O₅The polyol precursors of PG500, 501, 503, 504, 506 and PI-88 (2mg each) were dried for 3 days. Pour into each vial 1.77mg (2.0mCi)³⁵SO₃Pyridine complex with 2mg SO₃·Me₃Stock solution of N in 300. mu.L of anhydrous DMF (Aldrich, redried using freshly started 3A molecular sieves) was 50. mu.L. To the SO₃A further 600. mu.L of anhydrous DMF was added to the vial and distributed to each sample vial. The sample was heated to 60 ° for 66 hours. Adding SO to each vessel₃·Me₃N (14mg in 300 μ L anhydrous DMF) and the resulting solution was heated to 60 ° overnight. The vial was cooled to room temperature and stored at-80 ℃ awaiting purification.

By adding Na₂CO₃Each sample was quenched (saturated aqueous solution adjusted to pH 8-9), evaporated to dryness and treated with SEC (Biogel P2, 2.6 × 90cm, flow rate 30 mL/hour, 5 min/part.) fractions containing the desired material were examined using a Geiger-Muller counter and then DMB testing with CE.

Table 2: summary of radiolabelling assay results

Pharmacokinetic Studies

Male Sprague Dawley rats (250-350g) were used. The animals were allowed free access to food and water before and during the trial, during which time they remained unconstrained in the metabolic trial cages. With isofluraneRats were anesthetized. A catheter was inserted in the external jugular vein through an incision in the neck and subcutaneously passed to a second incision in the skin of the back (near the midline of the scapula). This was then removed from the abdomen under the protection of a light metal spring. The incision was closed and the spring was secured to the skin with Michel sutures so that the rat had the maximum range of motion. The animals were carefully monitored during recovery (1-4 hours).

Stock dose solutions were prepared by mixing appropriate amounts of unlabeled and radioisotope labeled drugs (dissolved in phosphate buffered saline) to give a total drug concentration of 1.25 mg/mL. All doses were administered as 2.5mg/kg bolus i.v. injections in a dose volume of 2 mL/kg. The total amount of radioactivity administered to each rat was 0.5-10 μ Ci. The dose level used in this study was 10-fold lower than the non-effective dose previously established for acute toxicity of PI-88. Before and 5, 15, 30, 45 minutes after administration of the dose, and 1, 1.5,2, 4, 8, 12, 24, 36 andblood samples (. about.250. mu.L) were collected for 48 hours. The blood sample was immediately centrifuged and the plasma was collected. At the completion of the test, the lethal excess of pentobarbital anesthetic IV was passedThe animals were sacrificed. Urine was collected from each animal at intervals of 0-12h, 12-24h, and 24-48h after dose administration. And cage washes (-15 mL deionized water) were collected. At the end of the experiment, the bladder contents of each animal were withdrawn and added to the 24-48h faeces. Feces were collected at the same time intervals as the urine.

Aliquots of plasma (100 μ L), urine and cage washes (500 μ L) were transferred directly to 6mL polypropylene scintillation vials for determination of radioactivity. Feces collected during each cycle (from the administration of each compound to one animal) were weighed and homogenized in 4 volumes of deionized water using a mechanical homogenizer. Approximately 1g (accurately weighed) of this slurry was transferred to a 20mL glass scintillation vial, 2mL of tissue lysing agent was added and the vial was capped and incubated at 60 ℃ for at least 24 hours. Radioactivity was measured after mixing the samples with a Packard Ultima gold liquid scintillation counting cocktail (2.0mL for plasma and dose, 5.0mL for urine and cage wash, 10mL for feces). The counts were performed on a Packard Tr-Carb liquid scintillation counter. Any result less than three times the background is considered to be less than the lower limit of the quantity and is not used in the calculation. Plasma, urine and cage washes were calculated as triplicates within 5 days of collection and no correction for radiochemical decay was made. Stool was processed as a batch at the completion of the study and counts from these samples were corrected for radiochemical decay. Plasma pharmacokinetic parameters were calculated using PK solutions2.0 software (Summit Research Services, Ohio) and are listed in table 3.

The results, listed in table 1, indicate that a wide range of compounds encompassed by the present invention have heparanase inhibitory activity and have strong affinity for GAG-binding growth factors and can therefore act as modulators of the activity of such factors in a similar manner to PI-88. In addition, the compounds have antiviral activity similar to that of PI-88. The results presented in Table 3 demonstrate that the compounds have altered pharmacokinetic properties compared to PI-88.

The above-described embodiments are merely illustrative of the principles of the present invention and various modifications and variations will be apparent to those skilled in the art. The invention is capable of embodiments and of being practiced and carried out in various ways and in other embodiments. It is also to be understood that the terminology used herein is for the purpose of description and should not be regarded as limiting.

The term "comprises" and variations of the term, such as "comprises" or "comprising," are used herein to connote inclusions of states or integers of states but not to exclude any other integers or any other integers, unless a specific interpretation of the term is required in the context or use.

Any reference to publications cited in this specification is not an admission that the disclosures form part of the common general knowledge in australia.

Reference to the literature

[1]Parish,C.R.;Freeman,C.;Brown,K.J.;Francis,D.J.;Cowden,W.B.CancerRes.1999,59,3433。

[2]Parish,C.R.;Cowden,W.B.6,143,730,2000。

[3]Iversen,P.O.;Sorenson,D.R.;Benestad,H.B.Leukemia2002,16,376。

[4]Ferro,V.;Don,R.Australas.Biotechnol.2003,13,38。

[5]Cochran,S.;Li,C.;Fairweather,J.K.;Kett,W.C.;Coombe,D.R.;Ferro,V.JMed.Chem.2003,46,4601。

[6]Vlodavsky,I.;Friedmann,Y.J.Clin.Invest.2001,108,341。

[7]Parish,C.R.;Freeman,C.;Hulett,M.D.Biochim.Biophys.Acta2001,1471,M99。

[8]Wall,D.;Douglas,S.;Ferro,V.;Cowden,W.;Parish,C.Thromb.Res.2001,103,325。

[9]Demir,M.;Iqbal,O.;Hoppensteadt,D.A.;Piccolo,P.;Ahmad,S.;Schultz,C.L.;Linhardt,R.J.;Fareed,J.Clin.Appl.Thromb.Hemost.2001,7,131。

[10]Hembrough,T.A.;Ruiz,J.F.;Papathanassiu,A.E.;Green,S.J.;Strickland,D.K.J.Biol.Chem.2001,276,12241。

[11]Amirkhosravi,A.;Meyer,T.;Chang,J.Y.;Amaya,M.;Siddiqui,F.;Desai,H.;Francis,J.L.Thromb.Haemost.2002,87,930。

[12]Francis,D.J.;Parish,C.R.;McGarry,M.;Santiago,F.S.;Lowe,H.C.;Brown,K.J.;Bingley,J.A.;Hayward,I.P.;Cowden,W.B.;Campbell,J.H.;Campbell,G.R.;Chesterman,C.N.;Khachigian,L.M.Circ.Res.2003,92,e70。

[13]Nyberg,K.;Ekblad,M.;T.;Freeman,C.;Parish,C.R.;Ferro,V.;Trybala,E.Antiviral Res.2004,63,15。

[14]Levidiotis,V.;Freeman,C.;Punler,M.;Martinello,P.;Creese,B.;Ferro,V.;van der Vlag,J.;Berden,J.H.M.;Parish,C.R.;Power,D.A.J.Am.Soc.Nephrol.2004,15,2882。

[15]Ferro,V.;Li,C.;Fewings,K.;Palermo,M.C.;Linhardt,R.J.;Toida,T.Carbohydr.Res.2002,337,139。

[16]Yu,G.;Gunay,N.S.;Linhardt,R.J.;Toida,T.;Fareed,J.;Hoppensteadt,D.A.;Shadid,H.;Ferro,V.;Li,C.;Fewings,K.;Palermo,M.C.;Podger,D.Eur.J.Med.Chem.2002,37,783。

[17]Ferro,V.;Fewings,K.;Palermo,M.C.;Li,C.Carbohydr.Res.2001,332,183。

[18]Parolis,L.A.S.;Parolis,H.;Kenne,L.;Meldal,M.;Bock,K.Carbohydr.Res.1998,309,77。

[19]Gunay,N.S.;Linhardt,R.J.Planta Med.1999,65,301。

[20]Ferro,V.;Hammond,E.;Fairweather,J.K.Mini-Rev.Med.Chem.2004,4,159。

[21]Alban,S.;Franz,G.Biomacromolecules2001,2,354。

[22]Foxall,C.;Wei,Z.;Schaefer,M.E.;Casabonne,M.;Fugedi,P.;Peto,C.;Castellot,J.J.,Jr;Brandley,B.K.J.Cell.Physiol.1996,168,657。

[23]Fugedi,P.;Tyrrell,D.J.;Tressler,R.J.;Stack,R.J.;Ishihara,M.5,739,115,1998。

[24]Katsuraya,K.;Nakashima,H.;Yamamoto,N.;Uryu,T.Carbohydr.Res.1999,315,234。

[25]Wessel,H.P.Topics Curr.Chem.1997,187,215。

[26]Chen,L.;Kong,F.J.Carbohydr.Chem.2002,21,341。

[27]Mori,M.;Ito,Y.;Ogawa,T.Carbohydr.Res.1989,192,131。

[28]Kerekgyarto,J.;Kamerling,J.P.;Bouwstra,J.B.;Vliegenthart,J.F.;Liptak,A.Carbohydr.Res.1989,186,51。

[29]Jacobsen,S.Acta Chem.Scand.Ser.B,Org Chem.Biochem.1984,B38,157。

[30]Ogawa,T.;Sasajima.Carbohydr.Res.1981,93,53。

[31]Ogawa,T.;Sasajima.Carbohydr.Res.1981,97,205。

[32]Garegg,P.J.;Olsson,L.;Oscarson,S.Bioorg Med.Chem.1996,4,1867。

[33]Fairweather,J.K.;Karoli,T.;Ferro,V.Bioorg Med.Chem.2004,12,6063。

[34]Karlsson,R.;Roos,H.;L.;Persson,B.Methods1994,6,99。

[35]Gunalp,A.Proc.Soc.Exp.Biol.Med.1965,118,185。

[36]Holland,T.C.;Homa,F.L.;Marlin,S.D.;Levine,M.;Glorioso,J.JVirol.1984,52,566。

Claims (7)

1. A compound of the general formula:

X-[Y]_n-Z-UR^l

wherein:

x, Y and Z are each the same hexose monosaccharide unit having a radical UR bonded to each of the non-linked carbons X, Y and Z by a single or multiple bond, except having a radical UR bonded by a single or multiple bond^lOther than carbon-1 of monosaccharide Z;

n is an integer of 0 to 6;

each U is independently N or O;

each R is independently C_1-10Alkyl, SO₃M or H, wherein M is any pharmaceutically acceptable cation;

R^lis acyl, PEG or PEG derivative, or R^lTogether with U being N₃；

Wherein at least 50% of said R groups are SO₃M。

2. A compound of the general formula:

wherein:

n is an integer of 0 to 6;

u is N or O;

each R is independently C_1-10Alkyl, SO₃M or H, wherein M is any pharmaceutically acceptable cation;

R^lis alkyl, aryl, acyl, PEG or PEG derivative, or R^lTogether with U being N₃；

Wherein at least 50% of said R groups are SO₃M。

3. A compound according to claim 1 or claim 2, wherein M is sodium.

4. A compound according to claim 1 or claim 2, wherein n is 3.

5. The compound of claim 2, wherein R^lIs n-octyl.

6. A compound according to claim 1 or claim 2, wherein R is^lIs an acetyl group.

7. A compound according to claim 1 or claim 2 wherein 70 to 100% of the R groups contain SO₃M。