KR20060129296A - Glycosaminoglycan (gag) mimetics - Google Patents

Abstract

The invention relates to compounds that are designed to mimic the structure of GAGs; methods for the preparation of the compounds; compositions comprising the compounds; and use of the compounds and compositions thereof for the anti-angiogenic, anti-metastatic, anti- inflammatory, anticoagulant, antithrombotic, and/or antimicrobial treatment of a mammalian subject.

Description

Glycosaminoglycan mimic {GLYCOSAMINOGLYCAN (GAG) MIMETICS}

The present invention relates to the field of compounds which mimic the structure of certain carbohydrates. In particular, the present invention relates to the field of glycosaminoglycans (GAG) mimetics.

Specifically, the present invention relates to compounds comprising one or more charged groups designed to mimic the structure of GAGs. The present invention also relates to methods of preparing the compounds, compositions comprising the compounds, uses and compositions of the compounds for the treatment of mammalian antiangiogenesis, antitransition, anti-inflammatory, anticoagulant, antithrombosis and / or antimicrobial. . Moreover, the present invention relates to the use of the compound and the composition in the treatment of a mammal having a condition treatable with such therapeutic agent.

GAG is a linear polyanionic polysaccharide that is produced in most animal cells and is generally found attached to the protein core (Refs. 1 and 2). GAGs are abundantly present (as proteoglycans) and are ejected by the cells to the cell surface and secreted into the extracellular matrix (ECM) [Ref. 3]. GAGs, particularly those belonging to the heparan sulfate (HS) family, mediate many physiological processes. For example, HS-GAG plays a major role in cell growth and development, angiogenesis, coagulation, cancer metastasis, cell adhesion, activation of growth factors, binding of cytokines and chemokines, and infection by bacteria and viruses. 4 to 6]. In recent years, there has been a dramatic increase in the list of proteins that interact with GAG, and the list continues to grow. Recent studies have shown that specific sequences in extracellular GAGs affect important biological processes by specifically binding to important proteins.

A molecule that mimics the structure of a particular GAG (referred to as a "GAG mimetic") can bind to a GAG-binding protein and its biological activity, such as activation of AT-III by several pentasaccharides [Refs. 7 and 8]. Or fibroblast growth factor (FGF) activation by sucrose octasulfate (Ref. 9). Similarly, it is known that GAG mimetics can antagonize GAG binding to a target protein and thereby inhibit the biological or disease function of that protein. For example, anticancer agents developed to target HS-binding angiogenic growth factors include polysulfonated compounds [ref. 10], suramin and related suradistas [ref. 11], and sulfated oligosaccharides [ref. Documents 12 and 13 are included.

The present invention relates to novel small molecule GAG mimetics that bind to and mediate their function. This compound contains one or more negatively charged groups (preferably sulfo groups) to interact with the charged residues in the amount of the GAG-binding site of the target protein, and also contains one or more substituents for the binding site Interacts with other protein residues in and around it. An important and special feature of the compounds mentioned herein is that they have a lower molecular weight than the above polysulfated GAG mimetics such as sulfated oligosaccharides with less sulfo groups [Refs. 12 and 13]. Another important feature is that Kisilevsky is based on cyclic scaffolds (eg monosaccharides) with sulfo groups and other substituents placed in a specific and predefined arrangement around the ring. It is significantly different from the simple, randomly charged GAG mimetics described by reference [14]. HS-binding of compounds described herein, binding to angiogenic growth factors, is demonstrated through surface plasmon resonance (SPR) solution affinity analysis. In addition, the compounds have been shown to inhibit HS-mediated infection and cell-to-cell spread of cells by herpes simplex virus.

One aspect of the present invention is to provide various GAG mimetics using the Ugi reactions [Refs. 15 and 16]. The scope of application of mimicking GAGs with various structural variations of GAGs and the variety of ways in which individual charged structures are connected to each other or to other functional groups is described. As is apparent in the art, the functionalization of the cyclic scaffold is not limited to the Ugi response. For example, the use of a number of other reactions such as alkylation, acylation and ring addition are described.

Summary of the Invention

It is an object of the present invention to provide novel charged compounds which function as GAG mimetics.

It is a further object of the present invention to provide an efficient synthetic route for the preparation of the compounds of this invention.

According to a first aspect of the invention, there is provided a compound of formula (I)

Where

n is an integer from 0 to 2;

Z is N, N (O), O, S, S (O), S (O) ₂ , P, P (O), P (O) ₂ , Si, Si (O) or Si (O) ₂ ;

Each X is independently C, C (O), N, N (O), O, S, S (O), S (O) ₂ , P, P (O), P (O) ₂ , Si, Si (O) or Si (O) ₂ , or a bond;

Each of R ₁ to R ₅ is independently a bond or hydrogen; halogen; Straight chain, cyclic, branched, substituted, heterocyclic, heteroatom substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or heteroaryl; Phosphoryl groups such as phosphate, thiophosphate -OP (S) (OH) ₂ , phosphate ester -OP (O) (OR) ₂ , thiophosphate ester -OP (S) (OR) ₂ , phosphonate -OP (O) OHR, thiophosphonate -OP (S) OHR, substituted phosphonate -OP (O) OR ₁ R ₂ , substituted thiophosphonate -OP (S) OR ₁ R ₂ , -OP ( S) (OH) (SH) and cyclic phosphates; Other phosphorus containing compounds such as phosphoramidite-OP (OR) -NR ₁ R ₂ and phosphoramidate-OP (O) (OR) -NR ₁ R ₂ ; A sulfur-containing group, for example, -OS (O) (OH), -SH, -SR, -S (→ O) -R, S (O) 2 R, RO-S (O) 2 -, -O-SO 2 NH ₂ , —O—SO ₂ R ₁ R ₂ or sulfamide-NHSO ₂ NH ₂ ; Amino groups such as —NHR, —NR ₁ R ₂ , —NHAc, —NHCOR, —NH—O—COR, —NHSO ₃ , —NHSO ₂ R, —N (SO ₂ ) R ₂ , and / or amidino groups ( Examples: -NH-C (= NH) NH ₂ ) and / or a ureido group (eg -NH-CO-NR ₁ R ₂ ) or a thioureido group (eg -HC (S) -NH ₂ ); Another unit of structure I bonded at any position, wherein Z, X and R ₁ to R ₆ are as defined above; And a substructure based on a group of formula (II)

Where

Y is a bond, straight chain, cyclic, branched, substituted, heterocyclic, heteroatom substituted or unsubstituted alkyl; Straight chain, cyclic, branched, substituted, heterocyclic, heteroatom substituted or unsubstituted acyl; And aryl, substituted aryl, heteroaryl, each R ₇ to R ₁₁ is independently one or more structures according to formula (I) or a structure according to formula (II),

Provided that when Z is O and X is O or a single bond, then R ₁ to R ₅ are not all H or CH ₂ OH, and when Z is N and X is O or a single bond, then R ₁ to R ₆ are all Not H

According to a second aspect of the present invention, angiogenesis, metastasis, inflammation of a mammal comprising at least one compound according to the first aspect together with a pharmaceutically or veterinary acceptable carrier or diluent for said at least one compound. Pharmaceutical or veterinary compositions for the prevention or treatment of diseases caused by coagulation, clotting, thrombosis and / or microbial infection are provided.

According to a third aspect of the invention, the use of a compound according to the first aspect for the manufacture of a medicament for the prevention or treatment of diseases caused by angiogenesis, metastasis, inflammation, coagulation, thrombosis and / or microbial infection in a mammal. Is provided.

According to a fourth aspect of the invention, angiogenesis, metastasis, inflammation, coagulation, in a mammal comprising administering to a mammal an effective amount of a compound according to one or more first aspects or a composition comprising one or more such compounds, Methods of preventing or treating diseases caused by thrombosis and / or microbial infection are provided.

In another aspect of the present invention, a method of synthesizing a compound according to the first aspect as defined above is provided.

Additionally for the compounds of the first aspect, unless otherwise specified, alkyl, aryl and other substituents are used according to the meanings commonly used in the art. For example, alkyl and aryl groups generally have from 1 to 10 carbon atoms. Additionally, two of the R ₁ to R ₅ groups may be linked to one another to form a bicyclic structure, or the cyclic structure of Formula I may contain a double bond, ie two adjacent XR ₁ to XR ₅ groups may be a bond.

Preferred compounds of the present invention have a structure of Formulas III-VI as defined in Tables 1-4 below.

In order that the present invention may be more readily understood and practiced, one or more preferred embodiments will be described by way of examples.

The following abbreviations are used herein.

GAG Glycosaminoglycans

HS Heparan Sulfate

FGF fibroblast growth factor

aFGF acidic fibroblast growth factor (or FGF-1)

bFGF basic fibroblast growth factor (or FGF-2)

VEGF Vascular Endothelial Growth Factor

SPR Surface Plasmon Resonance

HSV Herpes Simplex Virus

We have found that a wide range of compounds having GAG mimetic properties can be synthesized using a number of different methods as illustrated in the examples below. Such compounds have the purpose of preventing or treating diseases resulting from angiogenesis, metastasis, inflammation, microbial infection, coagulation or thrombosis in mammals. This use is due to the ability of compounds to mediate the activity of GAG-binding proteins responsible for disease processes.

GAG mimetics of the present invention can be synthesized using a number of different routes, including the Ugi reaction, as indicated above, and generally include sulfonation in the process.

Preferred compounds according to the first aspect of the invention as defined above include the compounds of formulas (I) and (II) and the compounds included in Tables 1 to 4 below.

As indicated above, the compounds according to the invention have a use in the prevention or treatment of diseases caused by angiogenesis, metastasis, inflammation, microbial infection, coagulation or thrombosis in mammals. In particular the compounds have a use in the treatment of such diseases in humans. Typically the compound is administered as a component of the pharmaceutical composition as described below.

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

Parenteral administration includes administration by the following routes: intravenous, skin or subcutaneous, nasal, intramuscular, intraocular, transdermal, intraperitoneal and topical routes. Topical administration includes skin, ocular, rectal and nasal administration as well as administration by inhalation or aerosol means. For intravenous, dermal or subcutaneous injection or injection at the site in need of treatment, the active ingredient may be in the form of a parenterally acceptable aqueous solution having no pyrogen and having adequate pH, isotonicity and stability. Those skilled in the art can readily prepare suitable solutions, such as solutions of the present compounds or derivatives thereof.

In addition to one or more of the present compounds and carriers or diluents, the compositions according to the invention further comprise pharmaceutically or veterinary acceptable additives, buffers, stabilizers, tonicity agents, preservatives or antioxidants or other substances known to those skilled in the art. It may include. It will be understood by those skilled in the art that such materials should be nontoxic and should not inhibit the efficacy of the present compounds. The exact type of optional additive may depend on the route of administration of the composition. That is, the composition may be administered orally or parenterally. With regard to buffers, aqueous compositions typically include such materials to keep the composition close to physiological pH, ie at least within the range of about 5.0 to 8.0 pH.

The compositions according to the invention may also comprise the active ingredient in addition to one or more compounds. Such ingredients are, in principle, selected for their efficacy as antiangiogenic agents, antimetastatic agents, anti-inflammatory agents, anticoagulants, antithrombotic agents, antimicrobial agents but may be selected according to their efficacy on the relevant condition.

Pharmaceutical or veterinary compositions according to the invention are administered to a subject in a prophylactically or therapeutically effective amount necessary for a particular condition. The substantial amount of one or more compounds administered by the composition, and the frequency and duration of administration, depend on the nature and the severity of the therapeutic or prophylactic condition desired. Therapeutic prescriptions, such as dosage, are within the role category of the healthcare practitioner or veterinarian responsible for the protection of the subject. Typically, however, compositions for administration to human subjects comprise about 0.01 to 100 mg per kg body weight, more preferably about 0.1 to 10 mg per kg body weight.

The compound may be included in the composition as a pharmaceutically or veterinary acceptable derivative thereof. As used herein, “derivatives” of compounds include salts, coordination complexes with metal ions (eg, Mn ²⁺ and Zn ²⁺ ), esters such as hydrolysable esters in vivo, free acids or free bases, hydrates or precursors Contains drugs. Compounds having acidic groups such as phosphates or sulfates may form salts with alkali or alkaline earth metals such as Na, K, Mg and Ca, and organic amines such as triethylamine and tris (2-hydroxyethyl) amine . Compounds having basic groups such as amines can also form salts by reaction with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid or tartaric acid. Compounds having both acidic and basic groups can form salts within themselves.

Esters can be formed using techniques known in the art, between the hydroxyl or carboxylic acid groups present in the compound and the appropriate carboxylic acid or alcohol reaction partner.

Prodrugs of the compounds of the invention may be converted to the parent compound in vivo or ex vivo. Typically, one or more biological activities of the parent compound can be inhibited in the prodrug form of the compound and activated by conversion of the prodrug to the parent compound or its metabolites. An example of a prodrug is a glycolipide derivative wherein at least one lipid moiety is provided as a substituent, which liberates the free form of the compound by cleavage reaction with an enzyme having phospholipase activity. Prodrugs of the compounds of the present invention include the use of protecting groups that can be removed in vivo to release the active compound or to inhibit loss of the drug. Suitable protecting groups are known in the art and include acetate groups.

As also indicated above, the compounds according to the invention have the use of the preparation of a medicament for the prevention or treatment of diseases caused by angiogenesis, metastasis, inflammation, coagulation, thrombosis and / or microbial infection in a mammal. Methods for the preparation of such medicaments are known in the art and include the methods used to prepare the pharmaceutical compositions.

Compounds within the scope of the present invention have been found to bind to growth factors. In particular, the compounds have affinity for aFGF, bFGF and VEGF. Thus, the compounds have use as anti-angiogenic agents, anti-metastatic agents and / or anti-inflammatory agents in the treatment of mammals, including humans. Uses of the compounds include the treatment of angiogenesis-dependent diseases such as angiogenesis and proliferative retinopathy associated with the growth of solid tumors, and the treatment of inflammatory diseases and disorders such as rheumatoid arthritis. The compounds can also be used for cardiovascular treatment by activating growth factors.

As further indicated above, the compounds of the present invention additionally have use as anticoagulants or antithrombotic agents. It can therefore be used for the prevention and treatment of a number of thrombosis and cardiovascular diseases, deep vein thrombosis, pulmonary embolism, thromboembolism, peripheral arterial thrombosis, unstable angina or myocardial infarction, best known as such diseases. Since the composition of charged amino acid compounds can be delivered orally, the present compounds are widely used but are a good alternative to warfarin, an oral anticoagulant with severe side effects.

The compounds of the present invention have also been found to inhibit viral infections and therefore have use as antiviral agents in the treatment or prevention of many viral infections. The compounds of the invention are particularly useful for the treatment or prevention of infections caused by pathogens using HS as an attachment / introduction receptor (e.g., HSV, HIV, Dengue virus, yellow fever virus, cytomegalovirus and hepatitis C virus). Suitable [Ref. 6]. Similarly, the compounds of the present invention are also suitable for the treatment or prevention of infections caused by nonviral microbial pathogens such as Plasmodium (malaria) using HS as attachment / introduction. Most notable is the inhibition of cell-to-cell proliferation of HSV-1 and HSV-2 by the compounds of the present invention.

Non-limiting examples of the compounds, their synthesis, and their biological activities, which broadly describe the invention, will be provided in conjunction with the tables and references briefly described below.

General process

General process for alkylation and deprotection of diols

Diol (1 equiv) in DMF was added dropwise to a cooled (0 ° C.), stirred suspension of NaH (5 equiv) pre-washed (hexane) in DMF. When the dropwise addition was completed, stirring was maintained (0 ° C. → room temperature, 20 minutes). The mixture was cooled (0 ° C., 5 minutes) and the alkyl halide (2 equiv) was added dropwise with continued stirring (0 ° C., room temperature, overnight). The mixture was cooled once more (0 ° C.) and MeOH (5 mL) was added with continued stirring (5 min). The solvent was evaporated and the residue was worked up (EtOAc) and purified by flash chromatography (TLC). This residue was co-distilled (2 × 10 mL MeCN). The crude mixture and p-TsOH.H ₂ O (50 mg) in MeOH / MeCN (1: 1) were heated to reflux (1 hour). The mixture was cooled (room temperature) and Et ₃ N (100 μL) was added before the solvent was evaporated. The residue was flash chromatographed (EtOAc / hexanes) to afford diols.

General process for sulfonation of alcohol

A mixture of alcohol and SO ₃ · trimethylamine (2 equivalents per hydroxyl group) in DMF was heated (60 ° C., Overnight). The cooled (room temperature) reaction mixture was treated with MeOH and then made basic (to pH> 10) by addition of Na ₂ CO ₃ (10% weight / weight). The mixture was filtered and the filtrate was evaporated and coevaporated (H ₂ O). Deacylation of the sulfated product was required, the crude product was taken up in water and 1M NaOH was added (2 equiv. Per acyl group). After deprotection was complete the product was transferred to the next step. Crude sulfated material in H ₂ O was subjected to size exclusion chromatography. Pure fractions were evaporated, co-evaporated (H ₂ O) and lyophilized (H ₂ O) to give the sulfated product. If necessary, after lyophilization, the product was passed through an ion exchange resin column (AG®-50W-X8, Na ⁺ type, 1 × 4 cm, deionized H ₂ O, 15 mL) to homogenize the product in the form of sodium salt. Switched. The collected solution was evaporated and lyophilized to give the final product as a colorless glass or white powder.

Size Exclusion Chromatography

Size exclusion chromatography (SEC) was performed on a Bio-Gel P-2, 5 × 100 cm column at a flow rate of 2.8 mL / min of 1.0 M NH ₄ HCO ₃ to collect a 2.8 minute fraction (7.8 mL). It was. The carbohydrate content of the fractions was analyzed by TLC (carbonization) and the multicharged species were analyzed by the dimethyl methylene blue test, followed by capillary electrophoresis (CE) to analyze the purity and lyophilized by collecting what was considered salt free.

In the presence of less sulfated by-products or other salt impurities (typically very small but frequently detected), LH20 SEC steps (2 × 95 cm, deionized water, 1.2 mL / min, 3.5 minutes per vial) are removed completely It was.

Dimethyl methylene blue test

A dimethyl methylene blue (DMB) reagent was prepared by dissolving 16 mg of DMB in 1 L of deionized water containing 3.04 g glycine and 2.37 g NaCl. The pH was adjusted to 3.0 by addition of 0.1 M HCl (95 ml). The stock solution was stored in a brown bottle at room temperature (this solution was stable for at least 3 months under these conditions).

A 96-well microtiter plate was loaded with 10 μL of fraction solution per well. 55 μL of DMB stock solution was added to each well. Immediate discoloration from blue to pink indicates that there is a multicharged species, ie sulfated product fraction.

General Process for NIS Glycosylation

Glycosyl acceptor (1 equiv), thioglycoside donor (1.1 equiv), 500 mg of freshly activated powder 3 μs molecular sieves and 10 mL of dry DCM were stirred at −20 ° C. for 20 minutes followed by addition of 1.3 equivalents of NIS and 1 drop of TfOH. Stirring was continued at −20 ° C. until the reaction was complete by TLC (about 1 hour) before adding 400 μL of Et ₃ N. Evaporation (vacuum) on silica gel and flash chromatography yielded the glycosylated product.

General Process for Ugi Four-Component Reaction

A solution of acid (1 equiv), amine (1 equiv), carbonyl compound (1 equiv) and isocyanide (1 equiv) in MeOH, MeOH-THF (various ratios) or CHCl ₃ was transferred to the reaction vial (final concentration) : 0.1 to 0.5 M). If D-glucononic acid is an acid component, it was added as a solid. For bis-acids, bis-amines, bis-aldehydes or bis-isocyanides the amount was 0.5 equivalents. The mixture was stirred or shaken at room temperature or 60 ° C. for 1 hour to 5 days. The reaction process was monitored by TLC. The mixture was evaporated and the residue was purified by flash chromatography or completely dried under high vacuum followed by direct peracetization and flash chromatography.

General process for acetylation of hydroxyl groups

The corresponding alcohol was dissolved in DCM-pyridine (15: 1 volume / volume, 0.15 M) containing DMAP (0.42 mol%). Acetic anhydride (2 equivalents per hydroxyl) was added and the mixture was stirred at rt overnight. The mixture was poured into ice cold 0.5M HCl and extracted with CHCl ₃ . The organic phase was separated and washed with cold 0.5M HCl (× 2), brine and dried (MgSO ₄ ). The solution was filtered and evaporated. The residue was purified by flash chromatography (gradient eluting with hexanes-EtOAc) to afford pure product.

General Process for Zemplen Deacetylation / Debenzoylation

A solution of acetate / benzoate in anhydrous MeOH (0.1M) was treated with a solution of methoxy sodium in MeOH (1.35M, 0.2-0.6 equiv). The mixture was stirred at rt for 1-3 h (monitored by TLC). The pH was adjusted to 6-7 by addition of acidic resin AG®-50W-X8 (H ⁺ type), the mixture was filtered and the resin washed with MeOH. The combined filtrate and washes were evaporated in vacuo and dried completely to afford polyol product.

General process for deprotection of benzyl esters via hydrocracking

To a solution of benzyl ether-protected compound (0.03 mmol) in MeOH or EtOH (2 mL) was added 5% Pd / C or 20% Pd (OH) ₂ (30 mg or greater) on activated carbon. The mixture was placed in a miniclave manufactured by Busier AG, Uster, Switzerland, and stirred for 2-10 hours under a hydrogen atmosphere (50 psi). Alternatively, the mixture was bubbled with hydrogen gas for 1 hour and then stirred for 1 to 5 days at room temperature under 1 atmosphere of hydrogen. The reaction was monitored by TLC (EtOAc or MeCN-water 10: 1). The mixture was filtered and washed with MeOH or EtOH. The filtrate was evaporated and dried under high vacuum, confirmed by ¹ H NMR, lyophilized and used directly for sulfonation.

Methylation of hydroxyl groups

The dry polyol was dissolved in anhydrous DMF under argon (0.04M) and stirred with NaH (60% suspension in mineral oil, 1.2 equiv. Per hydroxyl) for 1 h at room temperature. Indomethane (1.2 equiv per hydroxyl) was added and stirring continued overnight. MeOH was added and the mixture was evaporated on silica and purified by flash chromatography.

General process for reaction of the Huisgen ringside

Sulfated sugar azide was dissolved in water (0.75 M) and an acetylene solution (0.9 M, 1 equiv) in t-butanol was added. To this mixture was added copper (II) sulfate solution (0.3 M in water, 5 mol%) and sodium ascorbate solution (1 M in water, 20 mol%). The mixture was shaken overnight at room temperature in a mini shaker and column chromatography (silica 1 × 18 cm, gradient elution in the order of EtOAc-MeOH-H ₂ O 50: 2: 1, 20: 2: 1, 10: 2: 1) Purification with gave the corresponding triazole product.

Example 1: PG2038

Step a: Methyl 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-benzyl-β -D-glucopyranoside

Methyl 2,3,6-tri-O-benzyl-β-D-glucopyranoside [Ref. 17] (150 mg, 322 μmol), methyl 3,4,6-tri-O-acetyl-2-O-benzyl 200 mg of -1-thio-β-D-galactopyranoside [Ref. 18] (151 mg, 354 μmol) and 3 ′ molecular sieve were subjected to a general NIS glycosylation process using NIS 95 mg (422 μmol). Flash chromatography (gradient elution 20:80 to 25:75 EtOAc: hexanes) yielded 274 mg of partially deacetylated material. To this mixture was added 10 mL of DCM, 200 μL of acetic anhydride, 200 μL of Et ₃ N and 2 mg of DMAP, the solution was stirred for 1 hour and then evaporated and flash chromatography (gradient elution 25:75 to 30:70 EtOAc: hexanes) gave the title compound. 180 mg (66%) were obtained as a colorless glass.

Step b: Methyl 3,4,6-tri-O-acetyl-α-D-galactopyranosyl- (1 → 4) -β-D-glucopyranoside

Perlman catalyst (20 mg) and 20 μL of acetic acid were added to methyl 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-galactopyranosyl- (1 → 4) -2 in 10 mL of MeOH. To a solution of 90 mg (106 μmol) of, 3,6-tri-O-benzyl-β-D-glucopyranoside was added. Hydrogen atmosphere was applied with 3 vacuum purges and the suspension was stirred for 3 days. The residue after filtration, evaporation and co-evaporation with PhMe was flash chromatographed (gradient elution 100: 0 to 100: 3 EtOAc: MeOH) to give 47 mg (91%) of the title compound.

Step c: Methyl 2-O-sulfo-α-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-sulfo-β-D-glucopyranoside, tetrasodium salt ( PG2038)

The disaccharide (32.2 mg, 0.667 mmol) was subjected to standard sulfonation and deacetylation to give the title compound as a white foam (4.0 mg, 7.8%, 96% purity, CE: 7.18 min).

Example 2: PG2046 and PG2047

Step a: 2-azido-3,4,6-tri-O-benzoyl-2-deoxy-α-D-glucopyranosyl- (1 → 4) -1,6-anhydro-2-azido 2-deoxy-3-O-benzyl-β-D-glucopyranose

3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidadate in 1,2-DCE (5 mL) [Ref. 19] (201 mg, 0.453 mmol) and a solution of 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-β-D-glucopyranose [Ref. 20] (84 mg, 0.302 mmol) were activated molecular sieves. (3x powder 300 mg) was stirred in an argon atmosphere (room temperature, 30 minutes). The mixture was kept stirring (10 min), cooled (-20 ° C) and TBDMSOTf (21 μL, 0.091 mmol) was added dropwise and stirring was maintained (-20 ° C, 10 min). Et ₃ N (100 μL) was added and the mixture was filtered and evaporated. The residue was subjected to aqueous work up (EtOAc) and flash chromatography (10-40% EtOAc / hexanes) to give a pale yellow oil (130 mg). The residue was co-evaporated (2 × 10 mL MeCN) followed by a general procedure for gemplan deacetylation. The product was subjected to aqueous work up (EtOAc) to give a colorless oil (98 mg). The residue was coevaporated (2 × 10 mL MeCN). BzCl (210 μL, 1.81 mmol) was added to a solution of crude product (0.302 mmol, maximum) and pyridine (2 mL) in 1,2-DEC (3 mL) and the combined mixture was stirred (room temperature, overnight). The mixture was cooled (0 [deg.] C.) and MeOH (2 mL) was added with continued stirring (0 [deg.] C. → room temperature, 2 minutes), then the solvent was evaporated and co-evaporated (toluene). The residue was subjected to aqueous workup (EtOAc) and flash chromatography (10-30% EtOAc / hexanes) to afford two compounds.

First, the title compound was obtained as a colorless foam (101 mg, 46%, 3 steps).

Next, 2-azido-3,4,6-tri-O-benzoyl-2-deoxy-β-D-glucopyranosyl- (1 → 4) -1,6-anhydro-2-azido -2-Deoxy-3-O-benzyl-β-D-glucopyranose was obtained as a colorless oil (27 mg, 12%, 3 steps).

Step b: 2-deoxy-2-sulfamido-α-D-glucopyranosyl- (1 → 4) -1,6-anhydro-2-deoxy-2-sulfamido-3-O-benzyl- β-D-glucopyranose, 2 sodium salt (PG2046)

2-azido-3,4,6-tri-O-benzoyl-2-deoxy-α-D-glucopyranosyl- (1 → 4) -1,6-anhydro-2-azido-2- A mixture of deoxy-3-O-benzyl-β-D-glucopyranoside (127 μmol), Perman's catalyst (11 mg) and ammonium formate (300 mg) in 2: 1 MeOH: EtOAc (7 mL) was charged at 65 ° C. under argon. Heated to and terminated with TLC. The mixture was cooled to rt, filtered (0.2 μm) and evaporated. The crude amine was purified by SPE (300 mg C18 Waters cartridge, equilibrated with 5:95 MeOH: H ₂ O, eluting with 100: 0 MeOH: H ₂ O gradient) to give 53 mg (58%) of diamine. Without further purification, DMF (5 mL), SO ₃ · Me ₃ N (41 mg, 295 μmol) and NaHCO ₃ (40 mg, 475 μmol) were added to the diamine. The mixture was heated to 60 ° C. for 1 h, then cooled to rt and quenched with ice and Na ₂ CO ₃ (saturated aqueous solution). This suspension was stored overnight at −18 ° C. and the sample was filtered. The filtrate was evaporated. Water (1 mL) and NaOH (250 μL, 1M) were added and the solution was stirred overnight and then loaded directly onto SEC column (general process) to yield 22 mg (28% during 3 steps) of the title compound.

¹ influenced by solvent inhibition signal

Partially interrupted by ² H2 ¹

Step c: 2-deoxy-2-sulfamido-α-D-glucopyranosyl- (1 → 4) -1,6-anhydro-2-deoxy-2-sulfamido-β-D-glucopy Lanoside, disodium salt (PG2047)

2-deoxy-2-sulfamido-α-D-glucopyranosyl- (1 → 4) -1,6-anhydro-2-deoxy-2-sulfamido-3-O- in purified water (2 mL) Benzyl-β-D-glucopyranoside, disodium salt (12.9 mg, 20.8 μmol) and Perlman catalyst (5 mg) were left overnight at H ₂ 50 psi. The mixture was filtered and lyophilized to yield 10.7 mg (98%) of the title compound.

Example 3: PG2039 and PG2037

Step a: Methyl 3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-benzyl -β-D-glucopyranoside

Methyl 2,3,6-tri-O-benzyl-β-D-glucopyranoside (287 mg; 618 μmol), ethyl 3,4-di-O-acetyl-2,6-O-dibenzyl-1-thio A standard NIS glycosylation process using 181 mg (803 μmol, 1.3 equivalents) of NIS was performed for 302 mg (618 μmol) and 700 mg of 3 ′ molecular sieves for -β-D-galactopyranoside [Ref. 21]. Flash chromatography (2.5 × 20 cm, gradient elution EtOAc: hexanes 1: 5 to 3: 1) afforded the title compound as a colorless gum (176 mg, 32%).

Step b: Methyl 3,4-di-O-acetyl-α-D-galactopyranosyl- (1 → 4) -β-D-glucopyranoside

Methyl 3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl- (1 → 4) -2,3,6-, following standard debenzylation processes Tri-O-benzyl-β-D-glucopyranoside (88 mg, 98.8 μmol) was deprotected to afford the title compound as a colorless powder (42 mg, 97%).

Step c: Methyl 2,6-di-O-sulfo-α-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-sulfo-β-D-glucopyranoside, 5 Sodium Salt (PG2039)

42 mg (95.4 μmol) methyl 3,4-di-O-acetyl-α-D-galactopyranosyl- (1 → 4) -β-D-glucopyranoside following the standard sulfonation / deacetylation process Was converted to the white powder of the title compound (14.8 mg, 18%, CE: 6.12 min).

Step d: Methyl 2,6-di-O-benzyl-3,4-di-O-methyl-α-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-benzyl -β-D-glucopyranoside

Methyl 3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl- (1 → 4) -2,3, following standard deacetylation and methylation processes 6-Tri-O-benzyl-β-D-glucopyranoside (72 mg, 80.8 μmol) was converted to the title compound (62.7 mg, 93%) as a colorless gum.

Step e: Methyl 3,4-di-O-methyl-α-D-galactopyranosyl- (1 → 4) -β-D-glucopyranoside

Methyl 2,6-di-O-benzyl-3,4-di-O-methyl-α-D-galactopyranosyl- (1 → 4) -2,3,6-, following standard debenzylation processes Tri-O-benzyl-β-D-glucopyranoside (62.7 mg, 75.1 μmol) was deprotected to afford the title compound as a colorless gum (28 mg, 97%).

Step f: Methyl 3,4-di-O-methyl-2,6-di-O-sulfo-α-D-galactopyranosyl- (1 → 4) -2,3,6-tri-O-sulfo -β-D-glucopyranoside, 5 sodium salt (PG2037)

Title compound from methyl 3,4-di-O-methyl-α-D-galactopyranosyl- (1 → 4) -β-D-glucopyranoside (28 mg, 72.8 μmol) according to standard sulfonation processes (3.2 mg, 4.9%) was obtained.

Example 4: PG2053 and PG2042

Methyl 4-O-allyl-2,3-di-O-sulfo-α-L-rhamnoside, 2 sodium salts (PG2053)

General alkylation and deprotection of methyl 2,3-O-isopropylidene-α-L-lamnopyranoside [Ref. 22] (with allyl bromide) followed by a general sulfonation process to give the title compound as a colorless powder. Obtained. CE t _m = 10.48 min.

SO ₃ · trimethylamine was added in a reduced amount (1 equiv) to give exclusively methyl 4-O-allyl-2-O-sulfo-α-L-rhamside, sodium salt (PG2042). CE t _m > 25.00 min.

Example 5: PG2024

Methyl 4-O-benzyl-2,3-di-O-sulfo-α-L-rhamnoside, 2 sodium salts (PG2024)

Methyl 2,3-O-isopropylidene-α-L-lamnopyranoside (by benzyl bromide) was subjected to general alkylation and deprotection followed by a general sulfonation process to give the title compound as a colorless powder. CE t _m = 10.82 min.

Example 6: PG2054

Step a: Methyl 4-O-benzoyl-α-L-rhamnoside

Solution of methyl 2,3-O-isopropylidene-α-L-lamnopyranoside (200 mg, 920 μmol), benzoyl chloride (193 mg, 1.38 mmol) and Et ₃ N (364 μL, 2.76 mmol) in DCM (10 mL) Was stirred overnight. The resulting suspension (Et ₃ N · HCl precipitate) was diluted with DCM (50 mL), washed with NaHCO ₃ (saturated, aqueous solution), washed with water, washed with brine, dried (MgSO ₄ ) and evaporated. The residue was taken up in 50 mL of 1: 1 MeCN: H ₂ O and p-TsOH (10 mg) was added. The resulting solution was stirred to terminate the reaction (TLC, about 4 hours), evaporated and flash chromatography (1: 1 EtOAc: hexanes) to give 165 mg (64% over 2 steps) of the title compound as a colorless solid.

Step b: Methyl 4-O-benzoyl-2,3-di-O-sulfo-α-L-rhamnoside, disodium salt (PG2054)

Methyl 4-O-benzoyl-α-L-rhamnoside was subjected to a general sulfonation process to give the title compound as a colorless powder. CE t _m = 11.14 min.

Example 7: PG2041

Step a: 4,6-O-benzylidene-1,2-dihydro-D-glucal

A mixture of tri-O-acetyl-D-glucal (1.7 g, 6.25 mmol), AcOH (50 μL) and Pd (OH) ₂ / C (100 mg) in MeOH (15 mL) was heated vigorously overnight under H ₂ (1 atm). Stirred. The mixture was filtered, the solvent was evaporated and the residue was flash chromatographed (10-50% EtOAc / hexanes) to give tri-O-acetyl-1,2-dihydro-D-glucal as a colorless oil. This residue was co-evaporated (2 × 10 mL MeCN) and then subjected to gemplan deacetylation to give 1,2-dihydro-D-glucal as a colorless oil (825 mg, 89%). This residue was coevaporated (2 × 10 mL MeCN).

P-TsOH.H ₂ O (50 mg) in a solution of 1,2-dihydro-D-glucal (495 mg, 3.34 mmol) and α, α-dimethoxytoluene (753 μL, 5.01 mmol) in DMF (5 mL). Was added and the combined mixture was stirred (60 ° C., 1 h). Et ₃ N (100 μL) was added and the solvent was evaporated. The residue was flash chromatographed (0-5% MeOH / CHCl ₃ ) to afford the title compound as a colorless needle (503 mg, 64%).

Step b: 3-O-benzyl-4,6-di-O-sulfo-1,2-dihydro-D-glucal, 2 sodium salt (PG2041)

4,6-O-benzylidene-1,2-dihydro-D-glucal was subjected to the general procedure of alkylation (benzyl bromide), deprotection and sulfonation to afford the title compound as a colorless powder. CE t _m = 15.40 min.

Example 8: PG2030

Step a: 1,6-Anhydro-3-O-methyl-β-D-glucopyranose

p-toluenesulfonyl chloride (790 mg, 4.14 mmol) was added to a cold (0 ° C.) suspension of 3-O-methyl-D-glucopyranose (804 mg, 4.14 mmol) in pyridine (10 mL) and the reaction mixture was stirred. (0 ° C. → room temperature, 1.5 hours). Then Ac ₂ O (1.5 mL, 15 mmol) and N, N-dimethylaminopyridine (50 mg) were added and stirring continued (room temperature, 4 hours). The mixture was then cooled (0 ° C.) and MeOH (3 mL) was added and stirring continued (10 min) before the solvent was evaporated. The remaining oil was dissolved (EtOAc) and worked up to give tosylate as pale yellow oil (1.93 g). A mixture of crude tosylate (1.93 g) and NaOH (20 mL of 1.0 M, 20 mmol) in EtOH (20 mL) was heated (80 ° C., 1 h). The mixture was neutralized with acetic acid and the solvent was evaporated and coevaporated (toluene). The crude residue was treated with pyridine (10 mL), Ac ₂ O (5 mL) and N, N-dimethylaminopyridine (50 mg) and the combined mixture was stirred (room temperature, overnight). The mixture was treated with ice water (10 mL) and stirring continued (at room temperature, 3 h) followed by work up (EtOAc). The remaining oil was flash chromatographed (20-50% EtOAc / hexanes) to give 2,4-di-O-acetyl-1,6-anhydro-3-O-methyl-β-D-glucopyranose (a) and A non-separable mixture of 1,2,4-tri-O-acetyl-3-O-methyl-6-O-tosyl-α-D-glucopyranose (b) (ratio of 3: 1) was prepared as pale yellow oil ( 466 mg). The ratio was determined by the integration of the H-1 and 3-OMe signals observed in the ¹ H NMR spectrum.

The mixture of two compounds (456 mg) was subjected to gemplan deacetylation general procedure and the residue was subjected to flash chromatography (0 to 5% MeOH / EtOAc) to give the title compound as a colorless oil (162 mg, 33%, 3 steps). .

Step b: 1,6-Anhydro-4-O-benzyl-3-O-methyl-β-D-glucopyranose

A mixture of 1,6-anhydro-3-O-methyl-β-D-glucopyranose (155 mg, 0.88 mmol) and Bu ₂ SnO (241 mg, 0.97 mmol) in toluene (1.8 mL) was heated to reflux (water azeotropy). Mixing was removed) to bring the solution to half the original volume. The mixture was cooled (80 ° C.), BnBr (104 μL, 0.88 mmol) and Bu ₄ NBr (567 mg, 1.76 mmol) were added and stirring continued (overnight). The mixture was treated with MeOH (2 mL) and H ₂ O (1 mL) with continued stirring (10 min) and the solvent was evaporated. The residue was worked up (EtOAc) and flash chromatography (20-60%, EtOAc / hexanes) gave two compounds.

First, the title compound was obtained as a colorless oil (94 mg, 40%).

In addition, 1,6-anhydro-2-O-benzyl-3-O-methyl-β-D-glucopyranose was obtained as a colorless oil (91 mg, 39%).

Step c: 1,6-Anhydro-4-O-benzyl-3-O-methyl-2-O-sulfo-β-D-glucopyranose, sodium salt (PG2030)

1,6-Anhydro-4-O-benzyl-3-O-methyl-β-D-glucopyranose (84 mg, 0.32 mmol) was sulfonated according to the general procedure and flash chromatography (50/2/1 → 10/2/1 EtOAc / MeOH / H ₂ O) followed by SEC to give the title compound as a pale yellow powder (70 mg, 60%). CE t _m = 5.62 min.

Example 9: PG2012 and PG2013

Step a: N-benzyl-N- (cyclohexylacetamido) -1,2,3,4-tetra-O-acetyl-D-glucuronamide

According to the general procedure for the Ugi reaction, a solution of D-glucuronic acid (0.950 g, 4.89 mmol) and each of the following three reagents: benzylamine (2M in MeOH, 2.45 mL, 4.89 mmol), formaldehyde (2M in MeOH) , 2.45 mL, 4.89 mmol) and cyclohexyl isocyanide (1M in MeOH, 4.89 mL, 4.89 mmol) were added to the reaction vessel and the mixture was stirred at rt for 19 h. The volatiles were removed under reduced pressure and dried under high vacuum to afford N-benzyl-N- (cyclohexylacetamido) -D-glucuronamide as a yellow foam.

According to the general procedure for acetylation, the crude Ugi product was peracetylated and flash chromatography (gradient eluting at 1: 1 in hexanes-EtOAc 2: 1) to afford the title compound as pale yellow foam, 1.929 g, 66%. (Step 2, Rf = 0.37, hexane-EtOAc 1: 1). ¹ H NMR (CDCl ₃ , 400 MHz) was very complicated due to the presence of anomer and rotamer. The spectrum was not simplified after the temperature rose to 55 ° C. However, in pyridine-d ₆ at 100 ° C., each set of rotamers coalesced in a somewhat more simplified structure, so two anomers were clearly observed (α: β ratio = 69: 31).

1 H-NMR (pyridine-d 6, 400 MHz, δ 7.22, 100 ° C.): Only typical sugar protons appeared; Residual signals (except acetate singlets) appeared in complex and wide piles.

Step b: N-benzyl-N- (cyclohexylacetamido) -1,2,3,4-tetra-O-sulfo-α-D-glucuronamide, tetrasodium salt (PG2012) and N-benzyl- N- (cyclohexylacetamido) -1,2,3-tri-O-sulfo-α-D-glucuronamide, trisodium salt (PG2013)

According to the general procedure for deacetylation, the tetraacetate (0.441 g, 0.747 mmol) was deacetylated to give N-benzyl-N- (cyclohexylacetamido) -D-glucuronamide as pale yellow glass (0.316). g, 100%).

According to the general procedure for sulfonation, the tetrol (0.257 g, 0.608 mmol) was sulfonated (using sulfur trioxide pyridine complex, 60 ° C., 19 hours). The residue was co-evaporated with toluene and purified by flash chromatography (2.5 × 20 cm, eluted with EtOAc, MeCN, MeCN-Et ₃ N (10: 1), MeCN-Et ₃ NH ₂ O (110: 2: 11)). It was. Fractions were divided into two portions by TLC and CE. The less polar portion was re-purified by flash chromatography, LH20 (× 2) and ion exchange chromatography and lyophilized to give trisulfate PG2013 as a white downy powder (19.3 mg, 4.4%).

The polar portion was purified by LH20 column (× 2) and ion exchange column and then lyophilized to give tetrasulfate PG2012 as a milky white powder (7.6 mg, 1.5%).

Example 10: PG2064

Step a: 2- (N-acetyl-N-cyclohexyl) amino-N- (methyl 2,3,4-tri-O-benzyl-6-deoxy-α-D-mannopyranose-6-yl) Acetamide

According to the general procedure for the Ugi reaction, a solution of each of the following four reagents: acetic acid (2M in MeOH, 60 μL, 119 μmol), cyclohexylamine (2M in MeOH, 60 μL, 119 μmol), formaldehyde (2M in MeOH, 60 μL, 119 μmol) and methyl 2,3,4-tri-O-benzyl-6-deoxy-6-isocyano-α-D-mannopyranoside (0.721M in CHCl ₃ , 150μL, 108μmol) in a 4 mL reagent vial And the mixture was stirred at 60 ° C. for 19 h. The volatiles were removed under reduced pressure and flash chromatography (gradient eluting with hexanes-EtOAc 4: 1 to 1: 4) afforded the title compound as a colorless gum, 42 mg, 60% (Rf = 0.49, EtOAc).

Step b: 2- (N-acetyl-N-cyclohexyl) amino-N- (methyl 6-deoxy-2,3,4-tri-O-sulfo-α-D-mannopyranose-6-yl) Acetamide, trisodium salt (PG2064)

According to the general procedure for deprotection of benzyl ether, a mixture of said tribenzyl ether (42 mg, 0.065 mmol), 20% palladium on activated carbon (22 mg) in MeOH (2 mL) was stirred for 10 h under 50 psi of hydrogen atmosphere. Normal workup gave triol intermediates as colorless gums. According to the general procedure for sulfonation, triols were sulfonated (sulfur trioxide trimethylamine complex, 60 ° C., 19 hours) and the crude product was evaporated. The residue was purified via successive SEC (LH20 after Biogel P-2). The pure product was passed through an ion exchange column to convert to sodium salt and then lyophilized to give the title compound as a white downy powder (3.1 mg, 7.0%, 2 steps).

Example 11: PG2068

Step a

According to the general procedure for the Ugi reaction, a solution of monomethyl succinate (15.7 mg, 0.119 mmol) and each of the following three reagents: ethylamine (2M in MeOH, 60 μL, 119 μmol), formaldehyde (2M in MeOH, 60 μL, 119 μmol) and methyl 2,3,4-tri-O-benzyl-6-deoxy-6-isocyano-α-D-mannopyranoside (0.721 M in CHCl ₃ , 150 μL, 108 μmol) in a 2 mL sample vial And the mixture was stirred at rt for 19 h. The volatiles were removed under reduced pressure and flash chromatography (gradient eluting with hexanes-EtOAc 1: 1 to 1: 4 then EtOAc) gave the pure product as a colorless gum (46.8 mg, 65%).

Step b (PG2068)

According to the general procedure for deprotection of benzyl ether, a mixture of said tribenzyl ether (46.8 mg, 0.0706 mmol), 20% palladium on activated carbon (30 mg) in MeOH (3 mL) was stirred for 2 h under 50 psi of hydrogen atmosphere. Normal workup gave triol intermediates as colorless gums. In accordance with the general procedure for sulfonation, triols were sulfonated (sulfur trioxide trimethylamine complex, 60 ° C., 19 hours). The residue was dissolved in 1M NaOH (3 mL, 0.16 M). The mixture was stirred at rt overnight and concentrated under reduced pressure. The residue was purified via successive SEC (LH20 after bio-gel P-2). The pure product was passed through an ion exchange column to convert to sodium salt to give PG2068 as a white powder (4.9 mg, 9.8%, 2 steps).

Example 12: PG2075

Step a

3-chlorophenylacetic acid (223 mg, 1.307 mmol) was dissolved in MeCN (3 mL). Ammonia solution (28%, 0.26 mL, 3.8 mmol) was added. The mixture was stirred briefly and evaporated in vacuo. The residue was suspended in MeCN (3 mL), filtered and the white solid was washed with MeCN and lyophilized to give ammonium 3-chlorophenylacetate (0.195 g, 80%).

According to the general procedure for the Ugi reaction, a solution of the ammonium salt (22.5 mg, 0.120 mmol) and each of the following two reagents: formaldehyde (2M in MeOH, 60 μL, 119 μmol) and 2-isocyanoethyl 2,3, 4,6-Tetra-O-benzyl-α-D-mannopyranoside (0.762 M in CHCl ₃ , 157 μL, 120 μmol) was added to a 2 mL sample vial and the mixture was stirred at room temperature for 19 hours. The volatiles were removed under reduced pressure and the residue was purified by flash chromatography to give the product as a colorless gum (34.8 mg, 37%).

Step b (PG2075)

According to the general procedure for deprotection of benzyl ether, the tetrabenzyl ether (34.8 mg, 0.0439 mmol), 20% palladium on activated carbon (26 mg) in MeOH (2 mL) was stirred for 2 h under 50 psi of hydrogen atmosphere. Normal workup gave the tetrol intermediate as a colorless gum. According to the general procedure for sulfonation, the tetrol was sulfonated. The residue was purified via SEC (bio-gel P-2). Pure product was passed through an ion exchange column to convert to sodium salt and lyophilized to give PG2075 a white downy powder (10.6 mg, 28%, 2 steps).

Example 13: PG2014

Step a

2- (benzyl 3,4,6-tri-O-benzyl-α-D-mannopyranoside-2-yl) acetic acid (50 mg, 0.0835 mmol) and the following three reagents, according to the general procedure for the Ugi reaction. Respective solutions: Benzylamine (2M in MeOH, 41.8 μL, 0.0835 mmol), formaldehyde (2M in MeOH, 41.8 μL, 0.835 mmol) and 2-isocyanoethyl 2,3,4,6-tetra-O- Benzyl-α-D-mannopyranoside (0.415 M in MeOH, 201.4 μL, 0.0835 mmol) was added to a 2 mL sample vial and the mixture was stirred at rt for 19 h. General workup gave the product as a colorless gum (38.9 mg, 36%).

Phase b (PG2014)

According to the general procedure for deprotection of benzyl ether, a mixture of said octabenzyl ether (35MG, 0.0267 mmol) and 20% palladium on activated carbon (10 mg) in EtOH (4 mL) was stirred for 2 h under 50 psi of hydrogen atmosphere. Normal workup gave the octol intermediate as a colorless gum. According to the general procedure for sulfonation, the octol was sulfonated (sulfur trioxide trimethylamine complex, 60 ° C., 19 hours). The residue was purified via SEC (bio-gel P-2). The pure product was passed through an ion exchange column to convert to sodium salt to give PG2014 as a white powder (16.6 mg, 44%, 2 steps).

Example 14: PG2016

Step a

According to the general procedure for the Ugi reaction, a solution of trans-1,4-diaminocyclohexane (6.3 mg, 0.055 mmol) and each of the following three reagents: 2- (methyl 2,3,4-tri-O-benzyl α-D-mannopyranoside-6-yl) acetic acid (0.91 M in MeOH, 121 μL, 0.11 mmol), formaldehyde (2 M in MeOH, 55 μM, 0.11 mmol) and cyclohexyl isocyanide (1 M in MeOH, 110 μM) , 0.11 mmol) was added to a 2 mL sample vial and the mixture was stirred at room temperature for 5 days. The volatiles were removed under reduced pressure and flash chromatography (gradient eluting from hexanes-EtOAc 2: 1 to 1: 4) yielded the product as a colorless gum, 33.0 mg, 43% (Rf = 0.24, DCM-MeOH 95: 5 or Rf). = 0.48, MeCN-EtOAc 1: 1).

Phase b (PG2016)

According to the general procedure for the deprotection of benzyl ethers, a mixture of said hexabenzyl ether (33 mg, 0.0235 mmol) and 20% palladium on activated carbon (65 mg) in MeOH (2.8 mL) was stirred for 5 days under 1 atmosphere of hydrogen atmosphere. . Normal workup gave the hexol intermediate as a colorless gum. According to the general procedure for sulfonation, the hexol was sulfonated. The residue was purified by continuous column chromatography (ion exchange column after SEC on bio-gel P-2) to give PG2016 as a white powder (12.2 mg, 35%).

Example 15: PG2015

Step a

According to the general procedure for the Ugi reaction, a solution of 3,3-dimethylglutaric acid (7.1 mg, 0.0443 mmol) and each of the following three reagents: 3-aminopropyl 2,3,4,6-tetra-O- Benzyl-α-D-mannopyranoside (0.642M in MeOH, 138μL, 0.0886mmol), formaldehyde (2M in MeOH, 44.3μL, 0.0886mmol) and cyclohexyl isocyanide (1M in MeOH, 88.6μL, 0.0886 mmol) was placed in a 2 mL sample vial and the mixture was stirred at room temperature for 5 days. The volatiles were removed under reduced pressure and flash chromatography (gradient elution from hexane-EtOAc 2: 1 to 1: 4) gave the product as a colorless gum, 32.3 mg, 46% (Rf = 0.45, hexane-EtOAc 1: 3). Obtained.

Phase b (PG 2015)

According to the general procedure for deprotection of benzyl ether, the hexabenzyl ether (32.3 mg, 0.0202 mmol), 20% palladium on activated carbon (41 mg) in MeOH (2.8 mL) was stirred for 5 days under 1 atmosphere of hydrogen atmosphere. Normal workup gave the octol intermediate as a colorless gum. According to the general procedure for sulfonation, the octol was sulfonated. The residue was purified via SEC (bio-gel P-2) to give PG2015 as a white powder (12.6 mg, 37%).

Example 16: PG2155

Ethyl 2,6-di-O-benzyl-3,4-di-O-sulfo-β-D-galactopyranoside, disodium salt (PG2155)

The title compound is obtained as a colorless powder from ethyl 2,6-di-O-benzyl-3,4-di-O-sulfo-β-D-galactopyranoside [Ref. 23] via a general sulfonation process. It was.

Example 17: PG2163

Step a: Methyl 4-O-allyl-6-azido-6-deoxy-2,3-di-O-isopropylidene-α-D-mannopyranoside

A solution of methyl 6-azido-6-deoxy-α-D-mannopyranoside (311 mg, 1.419 mmol) in 2,2-dimethoxypropane (4.7 mL, 0.3 M) was added to (±) -campo-10. Treated with sulfonic acid (16 mg, 0.0709 mmol, 5 mol%). The mixture was stirred at rt for 1 h. TLC showed complete conversion to product (Rf = 0.40, EtOAc-hexane = 17: 83). The mixture was basified with addition of saturated Na ₂ CO ₃ (aq) and evaporated in vacuo. The residue was extracted with EtOAc (30 mL) and the EtOAc solution was washed with brine and dried (MgSO ₄ ). The gum was obtained by filtration and evaporation and it was once co-evaporated with toluene. The final colorless gum was dissolved in anhydrous DMF (3.5 mL, 0.4 M) and stirred with NaH (60% dispersion in mineral oil, 163 mg, 4.257 mmol, 3 equiv) for 1 hour. Allyl bromide (360 μL, 4.257 mmol, 3 equiv) was added and the mixture was further stirred at rt for 6 h, treated with methanol (1 mL) and evaporated to dryness. The residue was purified by silica column chromatography (2.5 x 18 cm, eluting with EtOAc in hexanes 1:10 to 1: 6) to afford the title compound as a colorless gum (0.281 mg, 66% for 2 steps).

Step b: Methyl 4-O-allyl-6-azido-6-deoxy-α-D-mannopyranoside

Methyl 4-O-allyl-6-azido-6-deoxy-2,3-di-O-isopropylidene-α-D-mannopyranoside (56 mg, 0.187 mmol) was added to MeCN-MeOH-H ₂ It was dissolved in O (3 mL, 3 mL and 0.2 mL, respectively) and treated with p-toluenesulfonic acid monohydrate (7 mg, 0.0374 mmol, 20 mol%). The mixture was stirred at rt for 5 h and triethylamine (0.4 mL) was added. The mixture was evaporated and the residue was purified by column chromatography (silica 1 × 18 cm, eluting with EtOAc-hexane 1: 6 to 2: 1) to give the product as a colorless waxy solid (34.8 mg, 72%).

Step c: Methyl 4-O-allyl-6-azido-2-O-benzyl-6-deoxy-2,3-di-O-sulfonato-α-D-mannopyranoside, 2 sodium salts ( PG2163)

Methyl 4-O-allyl-6-azido-6-deoxy-α-D-mannopyranoside was sulfonated according to standard procedures to yield the title compound as a white powder, 55 mg (89%). Rf = 0.20 (EtOAc-MeOH-H ₂ O = 10: 2: 1)

Example 18: PG2160, PG2161 and PG2173

Step a: Methyl 6-azido-6-deoxy-2,3-di-O-benzylidene-α-D-mannopyranoside

Methyl 6-azido-6-deoxy-α-D-mannopyranoside (1.011 g, 4.61 mmol) was dissolved in anhydrous DMF (9 mL) and acetonitrile (9 mL). Benzaldehyde dimethyl acetal (1.38 mL, 9.22 mmol, 2 equiv) and (±) -campo-10-sulfonic acid (214 mg, 0.922 mmol, 20 mol%) were added in this order. The mixture was stirred overnight at 60 ° C. (outside) under house vacuum and the volatiles were removed by rotary evaporator. The residue was loaded onto silica gel and purified by column (silica 2.5 × 20 cm, hexane-ethyl acetate 10: 1, 9: 1, 7: 1, 5: 1, 4: 1, 3: 1, 2: 1) Gradient elution). Fractions were combined in two parts. The less polar portions (Rf = 0.55 and 0.51, hexanes-EtOAc 3: 1) were mixtures of 4-acetal and the polar portions were mainly 4-OH products. The mixture was combined and evaporated. The residue was redissolved in dichloromethane (50 mL) and stirred with 1M ammonium chloride solution (50 mL). Less polar spots slowly disappeared and converted to a polar product. However, the conversion did not improve after 40 minutes. The dichloromethane phase was thus separated and stirred further with 0.5M HCl solution (50 mL) for 20 minutes. TLC no longer showed a change. The DCM phase was separated and washed with brine-1 M NaOH and dried (MgSO ₄ ). Dry DCM was filtered and evaporated and the residue was purified as above on a silica column to give the title compound as a colorless gummy solid (0.543 g, 38%, Rf = 0.29, EtOAc-hexane = 1: 3).

Step b: Methyl 6-azido-3-O-benzyl-6-deoxy-α-D-mannopyranoside and methyl 6-azido-2-O-benzyl-6-deoxy-α-D- Mannopyranoside

A solution of methyl 6-azido-6-deoxy-2,3-di-O-benzylidene-α-D-mannopyranoside (240 mg, 0.781 mmol) in DMF (7.8 mL, 0.1 M) was dissolved in sodium cyanobo. Treated with low hydride (589 mg, 9.37 mmol, 12 equiv) and 3 cc of molecular sieve (1 g). The mixture was stirred at room temperature for 20 minutes, then brought to 70 ° C. and TFA (0.361 mL, 4.686 mmol, 6 equiv) was added slowly. After addition, the mixture was stirred at 70 ° C. for 6 hours, cooled to 0 ° C. and basified by addition of solid Na ₂ CO ₃ . The cold mixture was filtered and the cake washed with EtOAc. The filtrate and washes were extracted once with saturated Na ₂ CO ₃ . Evaporation gave a gum which was column chromatographed (silica 2.5 × 18 cm, eluting 1: 1 in EtOAc-hexane 1: 4) to methyl 6-azido-3-O-benzyl-6-deoxy-α-D- Mannopyranoside (colorless gum, 64 mg, 26%, Rf = 0.45, EtOAc-hexane = 1: 1)

And methyl 6-azido-2-O-benzyl-6-deoxy-α-D-mannopyranoside (colorless waxy solid, 62 mg, 26%, Rf = 0.27, EtOAc-hexane = 1: 1 )

를 수득하였다.

Obtained.

Step c: Methyl 6-azido-2-O-benzyl-6-deoxy-2,3-di-O-sulfonato-α-D-mannopyranoside, disodium salt (PG2160)

Methyl 6-azido-2-O-benzyl-6-deoxy-α-D-mannopyranoside was sulfoned according to standard procedures to yield the title compound as a white powder, 64 mg, 59%.

Step d: Methyl 6-azido-3-O-benzyl-6-deoxy-2,4-di-O-sulfonato-α-D-mannopyranoside, disodium salt (PG2161)

Methyl 6-azido-3-O-benzyl-6-deoxy-α-D-mannopyranoside (64 mg) was sulfoned according to standard procedures to afford the title compound as a white powder, 58 mg, 66%. .

Step e: Methyl 6- [1 '-(4-phenyl) triazolyl] -2-O-benzyl-6-deoxy-2,3-di-O-sulfonato-α-D-mannopyranoside , 2 sodium salts (PG2173)

Whisgen Reaction General Process Using Methyl 6-azido-2-O-benzyl-6-deoxy-2,3-di-O-sulfonato-α-D-mannopyranoside, 2 Sodium Salt With Phenylacetylene The title compound was obtained through white powder, 6.8 mg, 67%, Rf = 0.34, EtOAc-MeOH-H ₂ O = 10: 2: 1.

Example 19: PG2170

Allyl 6-azido-2,3-O-disulfonato-6-deoxy-4-O- (1-naphthylmethyl) -α-D-mannopyranoside, disodium salt (2-naphthyl Containing 10% methyl isomer) (PG2170)

Prepared similarly to PG2163 starting with allyl 6-azido-6-deoxy-α-D-mannopyranoside, the title compound is prepared as a white powder, 87.5 mg, 87%, Rf = 0.28 (major), 0.22 (Miner), EtOAc-MeOH-H ₂ O = 10: 2: 1.

The source of minor isomers is a commercial 9: 1 mixture of 1- and 2-bromomethylnaphthylene used in step a.

Compounds were further synthesized by suitably modifying the synthesis described in Examples 1-19 above. Such additional compounds are included in the tables showing the biological test results of the compounds according to the invention.

Example 20

Biological Testing for Compounds

Way

1. Growth Factor Binding

The binding affinity of the ligand to the growth factor was measured using a solution affinity assay based on surface plasmon resonance (SPR). The principle of this analysis is that heparin immobilized on the surface of the sensor chip distinguishes between free and bound growth factors in the equilibrium solution of growth factors and ligands. By infusion of the solution, the free growth factor binds to immobilized heparin, which is detected as an increase in the SPR response and its concentration is measured. Reduction of free growth factor concentration as a function of ligand concentration allows the calculation of the dissociation constant K _d . It is important to note that because ligand binding to growth factors can only be detected when interaction occurs at the heparin binding site, it is not necessary to measure nonspecific binding at other positions on the protein. 1: 1 stoichiometry was assumed for all protein: ligand interactions.

The fabrication of heparin-coated sensor chips via the immobilization of biotinylated BSA-heparin on streptavidin-coated sensor chips has been described (Ref. 24). Heparin was also immobilized via aldehyde bonds with adipic acid dihydrazide or 1,4-diaminobutane. For each K _d measurement, a solution was prepared containing fixed concentrations of protein and various concentrations of ligands in buffer. Ligand binding to FGF-1 and VEGF was measured in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.0 mM EDTA and 0.005% (volume / volume) polysorbate 20), binding to FGF-2 Was measured in HBS-EP buffer containing 0.3 M NaCl (Ref. 24). Before injection, the sample was maintained at 4 ° C. to maximize protein stability. For each assay mixture, 50-200 μL of solution was injected at 5-40 μL / min and the relative binding reaction was measured. All surface bonding tests were conducted at 25 ° C. The surface was regenerated by injecting 40 μL of 4M NaCl at 40 μL / min and then 40 μL of buffer at 40 μL / min.

Sensorgram data was analyzed using BIA measurement software (BIAcore). The background sensorgram was removed from the experimental sensorgram to obtain a curve of specific binding and the baseline was subsequently adjusted to zero for all curves. Relative binding reaction for each injection was converted to free protein concentration using the following equation.

Wherein r is the relative binding reaction and r _m is the maximum binding reaction.

Established binding equilibrium in solution prior to injection was estimated to be 1: 1 stoichiometry. Therefore, the equilibrium can be expressed as follows.

In the above formula, P corresponds to a growth factor protein, L corresponds to a ligand, and P · L corresponds to a protein ligand complex.

The equilibrium equation is as follows.

In addition, the coupling equation [Ref. 24] can be expressed as follows.

The value of K _d is a fixed value calculated using a joining equation that is a function of the _sum of [P] versus [L]. If the K _d value is double calculated, the value is taken as the average of the double measurements. GAG mimetics that bind tightly to these growth factors exhibit biological responses in vivo (Ref. 24).

2. Antiviral Assay

For both types of herpes simplex virus (HSV), HSV-1 and HSV-2, selected compounds were described as described by Nyberg et al. [25] virus infection and cell-to-cell spread. The two assays for the inhibition of were tested. Monolayer cultures of African green monkey kidney cells (GMK AH1) cultured in 6-well cluster plates (Ref. 26) were used. The viral strains used were herpes simplex virus type 1 (HSV-1) KOS321 strain [Ref. 27] and HSV-2 strain 333 [Ref. 28].

In both assays the compounds were tested at 200 μM.

(i) In HSV infectivity assays, compounds were mixed with virus and incubated at room temperature for 10 minutes before the mixture was added to the cells and kept on the cells for 1 hour to enable virus attachment / introduction into cells. Thus, this assay looks at whether the compound of interest has a role in binding to viral particles and blocking attachment / introduction to cells. Inhibition results in a reduced number of viral plaques.

(ii) In the next assay, called HSV diffusion, by compound addition into cells after the virus attachment / introduction step has already taken place. This assay indicates whether the test compound has the ability to inhibit viral transfer (cell-to-cell spread) from infected cells to uninfected cells and additionally whether the compound has the ability to enter the cell and inhibit viral replication. . If the activity of the compound is reduced in the analysis of virus infectivity, but there is little activity in the virus spread assay, the compound is implicated to function by entering the cell and inhibiting the viral replication step. Inhibition results in a decrease in the size of the viral plaque.

The results (see Table 5) are expressed as a percentage of the control, ie the number (viral analysis) or size (diffusion analysis) of virus plaques present in the presence of the compound relative to the false-treated control (without compound).

result

The results of the test are shown in Tables 1-5.

The results in Tables 1-4 demonstrate that a wide range of compounds belonging to the present invention have a strong affinity for GAG-binding growth factors and therefore can act as modulators of their activity. The results shown in Table 5 demonstrate that the compound has truly in vivo activity.

The above embodiments merely illustrate the principles of the invention, and various variations and modifications can be readily made in the art. The invention can be implemented and practiced in many ways and in other aspects. Also, 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 (e.g., "comprising") are intended to encompass the integer (s) mentioned or other integers unless the context requires an exclusive interpretation of the term. It does not preclude (s).

References in documents cited herein do not mean that the disclosure constitutes common sense in Australia.

references

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[3] 20179369 Tomodova, S .; Woods, A .; Couchman, Jay. Tumova, S .; Woods, A .; Couchman, JR) Int. J. Biochem. Cell Biol. 2000, 32,269.

[4] capilla, child; Linhart, R. Capila, I .; Linhardt, RJ Angew. Chem. Int. Ed. 2002, 41, 391.

[5] Kasu, B .; Linda, B .; Lindahl, U. Adv. Carbohydr. Chem. Biochem. 2001, 57, 159.

[6] Liu, J .; Torp, S. Liu, J .; Thorp, SC Med. Res. Rev. 2002, 22, 1.

[7] Van Bokel, Mr. a. a; Van Boeckel, CAA; Petitou, M. Angew. Chem. Int. Ed. Engl. 1993, 32, 1671.

[8] pepitto, M .; Heraut, Jay. blood. (Petitou, M .; Heraut, JP; Bernat, A .; Driguez, PA; Duchaussoy, P .; Lormeau, JC; Herbert, JM) Nature 1999, 398, 417.

[9] Yes, rain. K. (Yeh, BK; Eliseenkova, AV; Plotnikov, AN; Green, D .; Pinnell, J .; Polat, T .; Gritli-Linde, A .; Linhardt, RJ; Mohammadi, M.) Mol. Cell. Biol. 2002, 22, 7184.

[10] Rickens, S. (Liekens, S .; Leali, D .; Neyts, J .; Esnouf, R .; Rusnati, M .; Dell Era, P .; Maudgal, PC; De Clercq, E .; Presta, M.) Mol. Pharmacol. 1999, 56, 204.

[11] Solar, F. (Sola, F .; Farao, M .; Pesenti, E .; Marsiglio, A .; Mongelli, N .; Grandi, M.) Cancer Chemother. Pharmacol. 1995, 36, 217.

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[21] Prepared similarly to Reference 18.

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Claims (14)

A compound of formula (I) Formula I

Where Each X is independently CH ₂ , C (O), N, O, S, S (O), S (O) ₂ or a bond; Each of R ₁ to R ₅ is independently a bond or hydrogen; halogen; Azide; C1 to C8 alkyl or alkenyl, aryl or heteroaryl, optionally further substituted with alkoxy, aryl, heteroaryl, or aryloxy group, -COOH, -S (O) ₂ OH, phosphate, carboxylate or tetrazolyl An R group as defined; -S (O) ₂ OH, -S (O) OH, -S (O) R, S (O) ₂ R, -S (O) ₂ NH ₂ , -S (O) ₂ OR, -S (O ) OR; -C (O) R; Phosphate, carboxylate or tetrazolyl; Heterocyclic groups, optionally substituted with alkyl or aryl groups, -CH ₂ NHC (O) R, -CH ₂ N (C (O) R) ₂ , -CH ₂ OR, wherein R is as defined above equivalence); A structure linked to different R ₁ to R ₅ to form a new cyclic group; And a substructure based on a group of formula (II), or a structure comprising a second unit according to formula (II), wherein each unit is independently connected via a “Y” group substituted by R ₇ to R ₁₀ , Provided that when R ₁ is —CH ₃ , —S (O) ₂ OH or —H, at least one of R ₂ to R ₅ is not —H or —S (O) ₂ OH; If no substructure of Formula (II) is present and neither of R ₁ to R ₅ forms an anhydrous bridge, then no more than two of R ₁ to R ₅ are —S (O) ₂ OH and the stereochemistry of Formula I is Or not galacto: Formula II

Where Y is H, R or -C (O) R, wherein R is as defined above; One or two groups of R ₇ to R ₁₁ are independently a structure according to formula (I). The method of claim 1, PG2024, PG2037, PG2173, PG2198 as described herein above. The method of claim 1, Compound of any one of the compounds of Table 1 to Table 4 of the present application. Induced by angiogenesis, metastasis, inflammation, coagulation, thrombosis and / or microbial infection in a mammal comprising at least one compound according to claim 1 and a pharmaceutically or veterinary acceptable carrier or diluent for said at least one compound Pharmaceutical or veterinary composition for the prevention or treatment of the disease being. The method of claim 4, wherein A composition further comprising a pharmaceutically or veterinary acceptable additive, buffer, stabilizer, tonicity agent, preservative or antioxidant. The method of claim 4, wherein Wherein said compound is present as an ester, free acid or base, hydrate or prodrug. Use of a compound according to claim 1 for the manufacture of a medicament for the prevention or treatment of diseases caused by angiogenesis, metastasis, inflammation, coagulation, thrombosis and / or microbial infection in a mammal. The method of claim 7, wherein The mammal is a human. Caused by angiogenesis, metastasis, inflammation, coagulation, thrombosis and / or microbial infection in a mammal comprising administering to the mammal an effective amount of at least one compound according to claim 1 or a composition comprising at least one said compound How to prevent or treat a disease. The method of claim 9, A method of preventing or treating a mammal as a human. The method of claim 9, A method for preventing or treating angiogenesis is angiogenesis caused by growth of proliferative retinopathy or solid tumor. The method of claim 9, A method for preventing or treating inflammation wherein the disease caused by inflammation is rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, allograft rejection or chronic asthma. The method of claim 9, A method for preventing or treating a disease caused by coagulation and / or thrombosis is deep vein thrombosis, pulmonary embolism, thrombosis stroke, peripheral arterial thrombosis, unstable angina or myocardial infarction. The method of claim 9, A method for preventing or treating a disease caused by viral infection, Herpes Simplex.