Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7016—Disaccharides, e.g. lactose, lactulose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/02—Acyclic radicals, not substituted by cyclic structures
  • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

• the oligosaccharide having this structure shows an increased resistance to enzymatic degradation and it negatively affects the growth of certain cancer cell lines.
• Another method allows to introduce a double bond into the structure of various glycosaminoglycans at positions 4 and 5 along the entire length of the chain, so that even polymers having a higher molecular weight can be effectively modified (Buffa R. et al. CZ305106, WO2014023272A1). These materials showed a selective negative effect on the cancer cells viability. Furthermore, an alkaline cleavage of the chondroitin sulphate benzyl ester in the mixture of DMF/EtOH/EtONa was disclosed (Gao, N. et al.: Carbohydrate Polymers, 127, 427-437, 2015). After de-esterification the resulting oligosaccharides showed better anticoagulant effects than a low-molecular-weight heparin.
• unsaturated oligosaccharides can be produced by enzymatic or chemical cleavage. Chemical cleavage is most often performed basicly and requires an activation, for example by converting an alcohol to an aldehyde or a carboxylic acid to its ester. Both oligosaccharides and polysaccharides can be used for cleavage.
• Hyaluronic acid or its sodium salt is an non-sulphated glycosaminoglycan composed of two repeating units of D -glucuronic acid and 7V-acetyl-D-glucosamine (Formula II).
• the molecular weight of the native hyaluronic acid can reach up to 5.10 6 g.mol' 1 .
• This polysaccharide forms an important part of connective tissues, skin and synovial fluid of joints and plays an important role in a number of biological processes such as hydration, cell differentiation and proteoglycans organization.
• Hyaluronic acid occurs naturally in biological systems, making it naturally biodegradable and biocompatible. Therefore, it is a suitable substrate for a wide range of biomedical applications.
• Hyaluronic acid is degraded in biological conditions by hydrolases and lyases.
• the products retain the structure according to the formula II, only the chain is shortened.
• hyaluronic acid is cleaved by lyase, a chain with a double bond is formed at the terminal saccharide at the non-reducing end (Formula III).
• the biological effects of oligomeric and polymeric hyaluronic acid differ significantly (Russo, R. I. C. et al.: International journal of cancer, 122, 1012-1018, 2008), it also depends on the way of hyaluronan cleavage (Jobe, K. L. et al.: Immunology letters, 89, 99-109, 2003).
• the reduction of aliphatic carboxylic acids can be carried out by a two-step synthesis, where in the first step the carboxyl group is activated by means oxalyl dichloride in N,N-dimethylformamide, tetrahydrofuran, dichloromethane or acetonitrile at -78 to +20 °C , and in the second step the activated carboxyl group is reduced with lithium tri-(tert-butoxy)aluminum hydride in tetrahydrofuran, dichloromethane and acetonitrile at 20 °C for 1.5 hours (Chany A.-C. et al.: Organic and Biomolecular Chemistry, 13, 35, 9190 - 9193, 2015).
• protic solvents Even in the reduction of carboxylic acids with aluminum-based hydrides, the use of protic solvents is unsuitable due to the rapid reaction of aluminum hydrides with the hydrogen of the solvent to form molecular hydrogen. Reductions proceed efficiently in aprotic solvents such as tetrahydrofuran, dichloromethane or acetonitrile.
• NaBEU has been used to reduce a wide range of carboxylic acids after in situ activation with benzotriazole-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate.
• the reaction proceeds rapidly under mild conditions in tetrahydrofuran with the addition of N,N- diisopropyl ethylamine to form high-yield alcohols (McGeary R. P.: Tetrahedron Letters, 39, 3319-3322, 1998).
• Reduction of the conjugate acid, in this case cinnamic acid results in an 18% reduction of the double bond.
• the carboxyl groups of the tetrasaccharide esterified to methyl esters, which also contain unprotected hydroxyl groups, can be reduced with NaBEU in methanol. Reduction to the corresponding alcohol proceeds for 1 hour at 20 °C. (DAcquarica I. et al.: Tetrahedron, 58, 51, 10127-10136, 2002).
• Aliphatic carboxylic acids containing ethers and acetamides can be reduced in water using an enzyme from Gloeosporium olivarum at 27 °C.
• the reaction time of 768 hours is extremely long, while racemization of the alpha position of the reduced carbonyl was also observed (TsudaY. et al.: Chemical and Pharmaceutical Bulletin, 33, 5, 1955-1960, 1985).
• the reduction of the conjugated double bond can proceed under the conditions used for the reduction of esters. In some cases, this reaction may be done on purpose, but often it is not desirable.
• unsaturated hyaluronic acid esters can be prepared by cleavage of the polymer in dimethyl sulfoxide in the presence of nitrogenous bases, since hyaluronic acid oligosaccharides in a basic environment undergo decomposition at an elevated temperature and cleave N-acetylglucosamine from the reducing end (Muckenschnabel, I. et al.: Cancer Letters, 1998, 131, 13-20).
• DMSO/water Example 8
• DMSO/methanol Example 9
• the oligosaccharide thus modified shows an increased resistance to enzymatic degradation and negatively affects the growth of certain cancer cell lines.
• the invention relates to a method of production of the oligosaccharide according to the structural formula I, where in the first step the carboxyl group of hyaluronic acid is alkylated to an ester, in the second step the ester is cleaved to form a double bond in positions 4 and 5 of the cycle, and in the third step the ester groups and the terminal anomeric center are reduced.
• the oligosaccharide according to the structural formula I can also be prepared by another method according to the invention, where, in the first step, hyaluronic acid is enzymatically cleaved using a lyase to an unsaturated oligosaccharide with a double bond in positions 4 and 5 of the cycle; in the second step, the unsaturated oligosaccharide is alkylated to an ester, and in the third step the ester groups and the terminal anomeric center are reduced to form a primary alcohol.
• the starting material is hyaluronic acid having a molecular weight of up to 2,000 kg. mol' 1 , where in the first step it is alkylated on the carboxyl group to form an alkyl ester of polymeric hyaluronan, in the second step selective chemical cleavage of the alkylated polysaccharide is carried out to form an unsaturated oligosaccharide and in the third step the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols.
• the starting material is hyaluronic acid having a molecular weight of up to 2,000 kg.mol-1, where in the first step it is enzymatically cleaved to form unsaturated oligosaccharides, in the second step it is alkylated on the carboxyl group to form alkyl esters of oligosaccharides, and in the third step the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols.
• the carboxyl group of polymeric hyaluronic acid is alkylated using an alkylating agent, for example benzyl bromide, dimethyl sulphate or ethyl iodide, in dimethyl sulfoxide in the presence of a base, for example diisopropyl ethylamine or triethylamine, to form an alkyl ester.
• an alkylating agent for example benzyl bromide, dimethyl sulphate or ethyl iodide
• a base for example diisopropyl ethylamine or triethylamine
• the molar amount of the base is preferably in the range from 1.7 to 6 equivalents and the molar amount of the alkylating agent is preferably in the range from 1.5 to 5 equivalents relative to the hyaluronic acid disaccharide.
• the initial hyaluronic acid can have a molecular weight in the range from 10 to 2,000 kg. mol' 1 .
• the concentration of hyaluronic acid in DMSO is preferably in the range from 0.3 to 10% by weight.
• a selective chemical cleavage of the solution of esterified hyaluronic acid in DMSO proceeds at a temperature in the range from 20 to 80 °C, preferably with heating, for 1 to 116 hours, in the presence of a base, for example tri ethylamine, N-methylmorpholine, or diisopropylethylamine, preferably in the range from 2 to 20 equivalents relative to the hyaluronic acid disaccharide.
• a base for example tri ethylamine, N-methylmorpholine, or diisopropylethylamine
• the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols.
• sodium borohydride NaBPU
• sodium borohydride (NaBPU) is added in an amount of 1 to 20 equivalents relative to the hyaluronic acid disaccharide and the mixture is stirred for 1 to 140 hours at a temperature in the range from 0 to 70 °C.
• the native hyaluronic acid is converted to a DMSO-soluble form before the first step, alkylation, for example by conversion to an acidic form by an acidification step with catex, or by conversion of the hyaluronic acid to a salt with organic counterions (e.g., tetrabutyl ammonium).
• alkylation for example by conversion to an acidic form by an acidification step with catex, or by conversion of the hyaluronic acid to a salt with organic counterions (e.g., tetrabutyl ammonium).
• the polymeric hyaluronic acid is enzymatically cleaved by the enzyme hyaluronate lyase in water at 36 to 38 °C for 16 to 92 hours to form an unsaturated oligosaccharide.
• the starting hyaluronic acid can have a molecular weight in the range from 100 to 2,000 kg. mol' 1 .
• the lyase is preferably selected from the group comprising Streptococcus pneumoniae hyaluronan lyase (SpHyl) and Streptococcus pyogenes hyaluronan lyase (Hylpl) and the lyase activity is preferably in the range from 1.632 to 1.75 lU/mL.
• the cleavage can be complete (down to di saccharides), preferably using SpHyl, or partial (a mixture of oligosaccharides).
• the carboxyl groups of oligomeric hyaluronic acid are alkylated using an alkylating agent, for example dimethyl sulphate, ethyl iodide or benzyl bromide, in dimethylsulfoxide in the presence of a base, for example triethylamine or diisopropylethylamine, to form an alkyl ester.
• an alkylating agent for example dimethyl sulphate, ethyl iodide or benzyl bromide
• a base for example triethylamine or diisopropylethylamine
• This step proceeds preferably for 5 to 120 hours at a temperature in the range from 20 to 25 °C, where the molar amount of the alkylating agent is preferably in the range from 1.2 to 3 equivalents relative to the hyaluronic acid disaccharide and the molar amount of the base is preferably 1.2 to 3 molar equivalent relative to the hyaluronic acid disaccharide.
• the concentration of the hyaluronic acid oligomer solution in DMSO is preferably in the range from 5 to 30% by weight.
• the esters of carboxylic acids and the anomeric end are reduced to form primary alcohols.
• sodium borohydride NaBHj
• sodium borohydride (NaBHj) is added in an amount of 1 to 20 equivalents relative to the disaccharide of hyaluronic acid and the mixture is stirred for 1 to 140 hours at a temperature in the range from 0 to 70 °C.
• the invention relates to the use of oligosaccharides composed of alternating glucose and N-acetyl glucosamine units, with an unsaturated glucose unit at the nonreducing end and a reduced anomeric end according to the structural formula (I).
• These materials negatively affect the growth of certain cancer cell lines, while not having a negative effect on the growth of normal cells. Furthermore, they show a high resistance to degradation by certain enzymes, which can increase the efficiency of their transport and prolong the duration of their effect. Therefore, they can be used in anti-cancer materials and compositions, for example, but not limited to, in the form of a solution for intravenous administration (by injection or infusion), or in the form of a solid tablet for oral administration.
• the unsaturated oligosaccharide according to the invention can therefore be used for the preparation of materials and compositions with an anti-cancer effect, preferably with an anti-cancer effect against colorectal cancer in humans.
• Fig. 1 Comparison of the rate of enzymatic degradation of the solution of the material prepared according to Example 36 (full line) and the native oligomer of hyaluronic acid (dotted line) after application of enzyme Streptoccus pneumoniae hyaluronate lyase (SpHyl).
• Fig. 2 Comparison of the rate of enzymatic degradation of the solution of the material prepared according to Example 36 (full line)) and the native oligomer of hyaluronic acid (dotted line) after application of bovine testicular hyaluronate hydrolase enzyme (BTH).
• BTH bovine testicular hyaluronate hydrolase enzyme
• Fig. 3 The effect of the materials prepared according to Examples 31, 33, 35 and 36 on fibroblasts NHDF viability.
• Fig. 4 The effect of the materials prepared according to Examples 31, 33, 35 and 36 on HT- 29 viability.
• Fig. 5 Comparison of the effect of native unsaturated HA oligosaccharides and the materials prepared according to Examples 31, 33, 35 and 36 on HT-29 viability.
• Hylpl Streptococcus pyogenes hyaluronate lyase
• SpHyl Streptococcus pneumoniae hyaluronate lyase
• DMEM Dulbecco's modified Eagle's medium
• FBS fetal bovine serum
• IP A isopropyl alcohol
• the term equivalent refers, unless otherwise indicated, to the repeating unit of the relevant polysaccharide or oligosaccharide, for example hyaluronic acid disaccharide.
• the percentages are given as percentages by weight unless otherwise stated.
• the molecular weight of the initial polysaccharides is weight average molecular weight, determined using the SECMALLS method.
• the lyophilizate was dissolved in dry DMSO (300 mL) under a nitrogen atmosphere. Then diisopropylethylamine (1.5 mL, 1.7 eq.) and dimethyl sulphate (0.375 mL, 1.5 eq.) were added and the reaction mixture was stirred 120 hours at 25 °C.
• Unsaturated disaccharide, prepared according to Example 14 (0.4 g), was dissolved in dry DMSO (1 mL). Then triethylamine (0.27 mL, 2 eq.) and ethyl iodide (0.24 mL, 3 eq.) were added and the reaction mixture was stirred at temperature 25 °C for 20 hours. Then acetic acid (0.1 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol.
• Unsaturated disaccharide, prepared according to Example 14 (0.8 g) was dissolved in dry DMSO (2 mL). Then diisopropylethylamine (0.7 mL, 2 eq.) and benzyl bromide (0.47 mL, 2 eq.) were added and the reaction mixture was stirred at temperature 20 °C for 16 hours. Then acetic acid (0.2 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol.
• a mixture of unsaturated oligosaccharides prepared according to Example 15 (0.1 g) was dissolved in dry DMSO (1 mL). Then diisopropyl ethylamine (0.083 mL, 2 eq.) and benzyl bromide (0.06 mL, 2 eq.) were added and the reaction mixture was stirred at temperature 25 °C for 120 hours. Then acetic acid (0.05 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol.
• Unsaturated disaccharide, prepared according to Example 14 (0.2 g) was dissolved in dry DMSO (1 mL). Then diisopropylethylamine (0.2 mL, 2 eq.) and dimethyl sulphate (0.1 mL, 2 eq.) were added and the reaction mixture was stirred at 20 °C for 5 hours. Then acetic acid (0.05 mL) was added and the reaction mixture was separated on a Cl 8 column in a mixture of water/methanol.
• a mixture of alkylated oligosaccharides (130 mg) prepared according to Example 10 was dissolved in DMSO (5 mL). Then NaBHj (130 mg, 10.5 eq.) was added and the reaction mixture was heated to 70 °C for 140 hours. Then acetic acid (5 mL, 20%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2-propanol (20 mL) and ethyl acetate (30 mL), washed and air dried.
• a mixture of alkylated oligosaccharides (252 mg) prepared according to Example 10 was dissolved in DMSO (5 mL). Then NaBEU (243 mg, 10 eq.) was added and the reaction mixture was heated to 50 °C for 140 hours. Then acetic acid (7.5 mL, 20%) was added and the reaction mixture was stirred for 30 minutes. The product was then precipitated by an addition of 2- propanol (30 mL), washed and air dried.
• the isolated alkylated disaccharide (103 mg) prepared according to Example 16 was suspended in MeOH (2 mL) and pyridine (0.08 mL). Then NaBHj (38 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2- propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.
• the isolated alkylated tetrasaccharide (203 mg) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBHj (76 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.
• the isolated alkylated hexasaccharide (204 mg) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBHj (81 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.
• the isolated alkylated octasaccharide (203 mg) prepared according to Example 11 was suspended in MeOH (4 mL) and pyridine (0.16 mL). Then NaBEU (76 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.15 mL, 99%) and 2-propanol (5 mL) were added. The precipitate was centrifuged, washed and air dried.
• the benzylated disaccharide (59 mg) prepared according to Example 18 was dissolved in MeOH (1 mL) and pyridine (0.04 mL). Then NaBHj (18 mg, 4 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.04 mL, 99%) and 2-propanol (4 mL) were added. The precipitate was centrifuged, washed and air dried.
• the methylated disaccharide (56 mg) prepared according to Example 20 was dissolved in MeOH (1 mL) and pyridine (0.04 mL). Then NaBHj (19 mg, 3.5 eq.) was added and the reaction mixture was stirred at 25 °C for 2 hours. Then acetic acid (0.04 mL, 99%) and 2- propanol (4 mL) were added. The precipitate was centrifuged, washed and air dried.
• NHDF viability (Fig. 3) represents the change in cell viability relative to the control at a given time - thus, an unaffected control corresponds to a value of 0; if the value is positive or negative up to -20% max., it is interpreted as the substance not having a cytotoxic effect.
• HT-29 viability (Fig. 4) represents the change in cell viability relative to the control at a given time - thus, an unaffected control corresponds to a value of 0; if the value is positive or negative up to -20% max., it is interpreted as the substance not having a cytotoxic effect.
• HT-29 viability (Fig. 5) represents the change in cell viability relative to the control at a given time - thus, an unaffected control corresponds to a value of 0; if the value is positive or negative up to -20% max., it is interpreted as the substance not having a cytotoxic effect.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• Pharmacology & Pharmacy (AREA)
• Wood Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Zoology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• General Chemical & Material Sciences (AREA)
• Microbiology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Epidemiology (AREA)
• Crystallography & Structural Chemistry (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=89224683

Family Applications (1)

Country Status (2)

Citations (1)

• 2022
  • 2022-11-10 CZ CZ2022-466A patent/CZ310437B6/cs unknown
• 2023
  • 2023-11-09 WO PCT/CZ2023/050077 patent/WO2024099487A1/en not_active Ceased

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events