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WO2018164662A1 - Esters de resvératrol - Google Patents

Esters de resvératrol Download PDF

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Publication number
WO2018164662A1
WO2018164662A1 PCT/US2017/020980 US2017020980W WO2018164662A1 WO 2018164662 A1 WO2018164662 A1 WO 2018164662A1 US 2017020980 W US2017020980 W US 2017020980W WO 2018164662 A1 WO2018164662 A1 WO 2018164662A1
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WO
WIPO (PCT)
Prior art keywords
resveratrol
ester
esters
amino
trihemiglutarate
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PCT/US2017/020980
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English (en)
Inventor
Mahmoud A. Elsohly
Waseem Gul
Jeptha N. COLE
Original Assignee
Elsohly Mahmoud A
Waseem Gul
Cole Jeptha N
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Application filed by Elsohly Mahmoud A, Waseem Gul, Cole Jeptha N filed Critical Elsohly Mahmoud A
Priority to PCT/US2017/020980 priority Critical patent/WO2018164662A1/fr
Publication of WO2018164662A1 publication Critical patent/WO2018164662A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/34—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C229/36—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton containing six-membered aromatic rings with at least one amino group and one carboxyl group bound to the same carbon atom of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/40—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/42—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton with carboxyl groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by saturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds

Definitions

  • Resveratrol (trans-3,4',5-trihydroxystilbene), a stilbenoid, is a natural polyphenol present in various plants, some food products, red wine and grapes. Resveratrol has the following chemical structure:
  • Resveratrol possesses anti-inflammatory, anti-carcinogenic and anti-oxidant properties, and has been extensively studied. Huge interest in resveratrol was created when it was discovered that it was able to activate the SIRT1 gene, a gene implicated in the life span extension associated with calorie-restricted diets.
  • resveratrol causes the over- expression of matrix metalloproteinase-9 (MMP-9), interleukin-8 (IL-8) and SIRT1 , and increases expression of epidermal growth factor receptor (EGFR) on the keratinocyte membrane and nucleus.
  • MMP-9 matrix metalloproteinase-9
  • IL-8 interleukin-8
  • SIRT1 epidermal growth factor receptor
  • SIRT1 may then promote differentiation, motility and proliferation of keratinocytes, and deacetylation and inactivation of p53 protein, inhibiting p53-dependent cell death from apoptosis in response to stress in human tenocytes (fibroblast-like tendon cells).
  • SIRT1 may induce nitric oxide (NO) production, which inhibits Class I HDAC 2 from blocking growth factors including epithelial growth factor, keratinocyte growth factor 2, fibroblast growth factor 10 (FGF-10) and insulin-like growth factor 1 (IGF-1).
  • SIRT1 may also decrease inflammation and apoptosis through a variety of mechanisms.
  • IL-8 has a direct and profound stimulatory effect on the migration of keratinocytes, which is likely via the PLC- ⁇ pathway. IL-8 may also recruit neutrophils.
  • MMP-9 degrades the Type IV collagen of the basement membrane.
  • EGFR may cause keratinocyte and fibroblast migration and may protect and repair tissue through nuclear DNA repair.
  • Resveratrol may also inhibit NF-KB-dependent pro-inflammatory and matrix- degrading gene products induced by IL- ⁇ and nicotinamide.
  • the invention is a resveratrol ester having the following structure:
  • R 1 , R 2 and R 3 are H or Each R 4 is independently a carbon chain of 2 to 4 carbon atoms comprising a terminal carboxylic acid moiety, a carbon chain of 1 to 5
  • the invention is a method of making a resveratrol ester, comprising forming the resveratrol ester from resveratrol.
  • the invention is a composition comprising a resveratrol ester and a pharmaceutically acceptable carrier.
  • the invention is a resveratrol ester selected from the group consisting of resveratrol hemimalonate, resveratrol hemisuccinate, resveratrol hemiglutarate, resveratrol 2-aminopropanoate, resveratrol 2-amino-3- methylbutanoate, resveratrol 2-amino-4-methylpentanoate, resveratrol 2-amino-3- methylpentanoate, resveratrol aminoethanoate, resveratrol 4-(4-aminophenyl)- butyrate, resveratrol 4-amino-butyrate, and resveratrol 6-amino-hexanoate.
  • resveratrol ester selected from the group consisting of resveratrol hemimalonate, resveratrol hemisuccinate, resveratrol hemiglutarate, res
  • the invention is resveratrol trihemiglutarate.
  • the invention is a method of reducing scar formation, comprising administering an effective amount of a composition comprising a resveratrol ester and a pharmaceutically acceptable carrier to a patient in need thereof.
  • the invention is a method of making a composition comprising a resveratrol ester and a pharmaceutically acceptable carrier.
  • the method does not include a solvent comprising alcohol.
  • Resveratrol esters include resveratrol esters of carboxylic acids, resveratrol esters of amino acids and amides thereof with dicarboxylic acids. Species of resveratrol esters contain the prefix mono-, di-, or tri- to indicate the number of ester linkages present in the resveratrol ester. The absence of the mono-, di-, or tri- prefix indicates a class containing the three species. For example, resveratrol
  • hemiglutarate refers to the class of resveratrol esters containing the three species resveratrol monohemiglutarate, resveratrol dihemiglutarate, and resveratrol trihemiglutarate.
  • a "resveratrol precursor” or a “resveratrol prodrug” is a compound that is converted to resveratrol by the body.
  • Hydrophill (or hydroxy-) refers to an— OH moiety.
  • Carboxylic acid (or carboxy-) refers to a compound with at least one— COOH moiety.
  • Dicarboxylic acid refers to a compound having two carboxylic acid moieties (—COOH).
  • Amino acid refers to a compound having an amine moiety (— NH2) and a carboxylic acid moiety (— COOH).
  • Amide refers to a compound with at least one— (CO) — moiety.
  • a “carbonyl carbon” is a carbon atom that is double-bonded to an oxygen atom.
  • esters linkage refers to the oxygen-carbonyl bond in an ester: o
  • FIG. 1 is the chemical structure of resveratrol trihemiglutarate.
  • FIG. 2 is a mass spectrum of resveratrol trihemiglutarate obtained by LC/MS.
  • FIG. 3A is a chromatogram of resveratrol obtained by HPLC in units of millivolts (mV) using an evaporative light scattering detector (ELSD).
  • FIG. 3B is a chromatogram of resveratrol obtained by HPLC in units of milli absorbance units (mAU) using a UV detector.
  • FIG. 4A is a chromatogram of resveratrol trihemiglutarate obtained by HPLC in units of millivolts (mV) using an ELSD.
  • FIG. 4B is a chromatogram of resveratrol trihemiglutarate obtained by HPLC in units of milli absorbance units (mAU) using a UV detector.
  • FIG. 5A is a microscopic image of untreated wound tissue.
  • FIG. 5B is a microscopic image of wound tissue that has been treated with a resveratrol ester.
  • FIG. 6 is a mass spectrum of resveratrol trihemisuccinate obtained by LC/MS.
  • FIG. 7 is a mass spectrum of resveratrol tri-alaninate-boc obtained by LC/MS.
  • FIG. 8 is a mass spectrum of resveratrol tri-alaninate HCI obtained by LC/MS.
  • FIG. 9 is a mass spectrum of resveratrol tri-valinate-boc obtained by LC/MS.
  • FIG. 10 is a mass spectrum of resveratrol di-valinate-boc obtained by LC/MS.
  • FIG. 11 is a mass spectrum of resveratrol mono-valinate-boc obtained by
  • FIG. 12 is a mass spectrum of resveratrol tri-valinate HCI obtained by LC/MS.
  • FIG. 13 is a mass spectrum of resveratrol tri-valinate-hemisuccinate obtained by LC/MS.
  • FIG. 14 is a mass spectrum of resveratrol tri-phenylalaninate obtained by
  • resveratrol such as into a wound
  • the delivery of resveratrol may be improved by administering a resveratrol precursor having greater water solubility than resveratrol.
  • a smaller amount of the resveratrol precursor may be used to deliver a therapeutically equivalent amount of resveratrol.
  • resveratrol precursor such as a resveratrol ester
  • a resveratrol precursor with sufficient water solubility to improve local administration and bioavailability of resveratrol, but not so water soluble so as to diffuse away from the application site.
  • resveratrol Another challenge presented by the low solubility of resveratrol is the difficulty in preparing aqueous compositions containing resveratrol.
  • preparation of an aqueous composition containing resveratrol involves a two-step process.
  • Resveratrol is first dissolved in an alcohol, such as ethanol. Next, the alcohol is dissolved in an alcohol, such as ethanol. Next, the alcohol is dissolved in an alcohol, such as ethanol.
  • resveratrol/alcohol solution is dissolved in water to form an aqueous composition.
  • this two-step process overcomes the problems presented by the low solubility of resveratrol in water, it is disfavored when the composition containing resveratrol is to be used in the reduction of scarring because alcohols are known fibrotic agents.
  • the amount of alcohol in the aqueous composition containing resveratrol may be reduced, but cannot be completely eliminated.
  • compositions containing resveratrol prepared by a process involving dissolution in alcohol will contain some amount of a fibrotic agent.
  • the present invention makes use of the discovery of resveratrol precursors with greater water solubility and greater bioavailability than resveratrol that are not so water soluble so as to diffuse away from the application site.
  • Resveratrol esters were identified as promising precursor candidates for resveratrol delivery because of the wide availability of esterases in vivo.
  • Applicants have surprisingly discovered that certain resveratrol esters possess unexpected and superior efficacy and bioavailability as compared to resveratrol.
  • the resveratrol esters were found to have increased water solubility while still allowing the resveratrol molecules to enter cells and provide the intended therapeutic benefits.
  • the esters chosen provide increased polarity without being so hydrophilic that the resveratrol precursors diffuse away from the site of application.
  • the present invention also makes use of the discovery of an improved method for producing aqueous compositions containing resveratrol precursors, such as resveratrol esters.
  • Resveratrol esters have increased water solubility as compared to resveratrol, which allows compositions containing resveratrol esters to be prepared without first dissolving the resveratrol esters in alcohol.
  • the method eliminates the introduction of alcohol, a known fibrotic agent, while also simplifying the production process.
  • Resveratrol esters may have an ester linkage at any of the three hydroxyl moieties on resveratrol. Resveratrol esters may be formed by any suitable chemical reaction, such as esterification with a dicarboxylic acid or esterification with an amino acid. Resveratrol esters include resveratrol with one, two, or three of the hydroxyl moieties modified by an ester linkage. Preferably, the resveratrol hydroxyl moieties have the same ester linkage when more than one resveratrol hydroxyl moiety is modified.
  • Resveratrol esters of carboxylic acids have an ester linkage between one or more of the resveratrol hydroxyl moieties oxygens and the carbonyl carbon from the carboxylic acid moiety.
  • the carboxylic acid used is a dicarboxylic acid.
  • Dicarboxylic acids are preferred because they retain a carboxylic acid moiety after esterification at the other carboxylic acid moiety. Retention of a carboxylic acid moiety increases the acidity of the resveratrol esters, which in turn increases the solubility of the resveratrol esters.
  • the dicarboxylic acid is a linear saturated dicarboxylic acid containing up to 5 carbon atoms.
  • Suitable dicarboxylic acids include malonic acid (propanedioic acid), succinic acid (butanedioic acid) and glutaric acid (pentanedioic acid).
  • a preferred dicarboxylic acid is glutaric acid.
  • esters of resveratrol with monocarboxylic acids have lower water solubility than resveratrol itself and are too lipophilic for use in resveratrol delivery.
  • Preferred resveratrol esters include hemimalonate [— (CO)(CH2)(CO)(OH)], hemisuccinate [— (CO)(CH 2 ) 2 (CO)(OH)] and hemiglutarate [— (CO)(CH 2 ) 3 (CO)(OH)].
  • a preferred hemiglutarate ester of resveratrol is resveratrol trihemiglutarate. The structure of resveratrol trihemiglutarate is shown in FIG. 1.
  • Resveratrol esters of dicarboxylic acids may be formulated as salts, for example, the sodium, potassium, calcium, or magnesium salts.
  • Resveratrol esters of amino acids have an ester linkage between one or more of the resveratrol hydroxyl moieties oxygens and the carbonyl carbon from the carboxylic acid moiety of the amino acid. If resveratrol esters of amino acids are formed by esterification, the amine moiety must be protected before the carboxylic acid moiety participates in esterification, such as with the te/t-butyloxycarbonyl protecting group (boc or t-boc). After esterification, the amine moiety may optionally be de-protected. Resveratrol esters of amino acids are often more stable than resveratrol esters of dicarboxylic acids. Preferably, the amino acid used has a low molecular weight.
  • Suitable natural amino acids include alanine (2-aminopropanoic acid), valine (2-amino-3-methylbutanoic acid), leucine (2-amino-4-methylpentanoic acid), isoleucine (2-amino-3-methylpentanoic acid), glycine (aminoethanoic acid) and phenylalanine (2-amino-3-phenylpropanoic acid).
  • Suitable non-natural amino acids include 4-(4-aminophenyl)-butyric acid, 4-amino-butyric acid and 6-amino-hexanoic acid.
  • a preferred amino acid is valine.
  • Preferred resveratrol esters formed from natural amino acids include 2-aminopropanoate (alaninate) [— (CO)(NH2)CHCH3], 2- amino-3-methylbutanoate (valinate) [— (CO)CH(NH 2 )CH(CH 3 )2], 2-amino-4- methylpentanoate (leucinate) [— (CO)CH(NH 2 )CH2CH(CH 3 )2], 2-amino-3- methylpentanoate (isoleucinate) [— (CO)CH(NH2)CH(CH3)CH 2 CH 3 ], aminoethanoate (glycinate) [— (CO)CH2NH2] and 2-amino-3-phenylpropanoate (phenylalaninate) [— (CO)(NH 2 )CHCH2C6H5].
  • Preferred resveratrol esters formed from non-natural amino acids include 4-(4-aminophenyl)-butyrate [— (CO)(CH2)3(C6H4) H2], 4-amino- butyrate [— (CO)(CH 2 )3NH 2 ], and 6-amino-hexanoate [— (CO)(CH 2 )5NH 2 ].
  • the resveratrol ester may be provided without removing the protecting group, such as resveratrol tri-alaninate-boc.
  • Resveratrol esters of amino acids may be formulated as salts, for example, the hydrochloride salt.
  • Resveratrol esters of amino acids also include resveratrol esters of amides.
  • Resveratrol esters of amides may be formed by reacting the amine moiety of a resveratrol ester of an amino acid with a dicarboxylic acid having a carbon chain of 3 or 4 carbon atoms.
  • Suitable dicarboxylic acids include malonic acid (propanedioic acid) and succinic acid (butanedioic acid).
  • Preferred resveratrol esters of amides include N-hemimalonate [— (CO)(CH2)(CO)(OH)] and N-hemisuccinate [—
  • Resveratrol esters of amides may be formulated as salts.
  • a resveratrol ester has the following general structure:
  • R 1 , R 2 , and R 3 may be a hydrogen atom (H) or .
  • Each R 4 is independently a carbon chain of 2 to 4 carbon atoms having a terminal carboxylic acid moiety, a o
  • R 5 is a carbon chain of 3 or 4 carbon atoms having a terminal carboxylic acid moiety. At least one of R 1 , R 2 , and R 3 is not H.
  • Each R 4 may be substituted or unsubstituted, saturated or unsaturated, and straight or branched. Preferably, each R 4 is unsubstituted, saturated and linear.
  • R 1 , R 2 , and R 3 may be the same, or may be different.
  • the resveratrol esters may optionally be formulated as salts.
  • compositions containing resveratrol esters are preferably prepared without first dissolving the resveratrol esters in alcohol.
  • Resveratrol esters may be dissolved in emulsifiers and solubilizers that do not contain alcohol.
  • Suitable solvents include emulsifiers and solubilizers in the KOLLIPHOR® portfolio produced by BASF.
  • a preferred solvent is KOLLIPHOR® ELP.
  • compositions containing resveratrol esters may optionally contain agents that do not materially affect the basic and novel characteristics of the resveratrol esters.
  • compositions containing resveratrol esters may optionally include agents such as stabilizers, preservatives or pH adjusters. If the compositions containing resveratrol esters are administered topically, the pH of the compositions must be carefully chosen to deliver the ester in its intended form without being irritating to the skin or tissue.
  • the pH of compositions containing resveratrol esters that are administered topically is 4.0 - 7.0 to closely match the pH of normal skin.
  • the resveratrol esters are present in a composition at a
  • the resveratrol esters are present in those compositions at a concentration of at most 1000 micromoles/liter. Examples include 7.5, 8.0, 9.0, 10, 12.5, 15, 16, 17, 18, 19, 20, 21 , 21.9, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 and 500 micromoles/liter.
  • Premeasured amounts of the compositions containing resveratrol esters may also be used. These are referred to as unit dosage forms, since each premeasured amount is intended to be used on a single patient for one or more application, all used at the same time. Examples include prefilled syringes, pouches, packets and tubes. Another example is a tube or dispenser which may be used to form foam of its contents just prior to application, for example by shaking or using a foaming agent. A self-foaming tablet, which forms foam when placed into water, could also be used. The volume of material present in these unit dosage forms may be 0.1 to 100 mL, or 1 to 50 mL, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30, 35, 40 and 45 mL
  • Example 1 Resveratrol trihemiglutarate synthesis
  • the following scheme depicts the process of preparing the trihemiglutarate ester of resveratrol:
  • This semi-solid gum was separated by chromatography on silica gel (60% ethyl acetate in hexanes with 0.1% trifloroacetic acid) and pure resveratrol trihemiglutarate (6) was obtained after combining the pure fractions (2.12 g).
  • FIG. 3A is a chromatogram of resveratrol (1) obtained using an ELSD.
  • FIG. 3B is a
  • FIG. 4A is a chromatogram of resveratrol trihemiglutarate (6) obtained using an ELSD.
  • FIG. 4B is a chromatogram of resveratrol trihemiglutarate (6) obtained using a UV detector.
  • Resveratrol was dissolved in tetrahydrofuran (THF). 4-dimethylaminopyridine (DMAP) was added to the solution while stirring. Succinic anhydride and
  • the mass of the resveratrol trihemisuccinate product was confirmed by LC/MS.
  • the mass spectrum is shown in FIG. 6.
  • Resveratrol was dissolved in dichloromethane (DCM). 4- dimethylaminopyridine (DMAP) was added to the solution while stirring.
  • DMAP dichloromethane
  • boc- -alanine hydroxide and dicyclohexylcarbodiimide (DCC) were dissolved in dichloromethane.
  • the resveratrol solution and the alanine solution were combined while stirring to produce crude resveratrol tri-alaninate-boc.
  • the crude resveratrol tri-alaninate-boc was dissolved in dichloromethane/ethyl acetate and loaded onto a silica gel column packed in hexanes.
  • Resveratrol tri-alaninate-boc was prepared according to Example 3. The
  • resveratrol tri-alaninate-boc was then dissolved in tetrahydrofuran (THF).
  • Hydrochloric acid (HCI) gas was bubbled through the solution at room temperature while stirring. A white precipitate was formed, resveratrol tri-alaninate HCI, and was filtered to produce a pure product.
  • the product was brought down with a step gradient beginning at 100% hexane (with 0.1 % trifluoroacetic acid (TFA)) and increasing ethyl acetate until reaching 60% hexane/40% ethyl acetate (with 0.1% TFA).
  • Fractions were collected and tested by thin layer chromatography (TLC) for purity. Optimal separation on TLC plates occurred in 40% ethyl acetate/60% hexane with 0.1 % TFA.
  • TLC showed three products: Resveratrol mono-valinate-boc, resveratrol di-valinate-boc, and resveratrol tri-valinate-boc. Similar fractions containing pure product were combined. Some fractions containing mixtures of products were ran on additional silica gel columns under the same conditions.
  • Resveratrol tri-valinate-boc was prepared according to Example 5. The
  • resveratrol tri-valinate-boc was then dissolved in tetrahydrofuran (THF).
  • Hydrochloric acid (HCI) gas was bubbled through the solution at room temperature while stirring. A white precipitate was formed, resveratrol tri-valinate HCI, and was filtered to produce a pure product.
  • Resveratrol tri-valinate was dissolved in dichloromethane (DCM). 4- dimethylaminopyridine (DMAP) was added to the solution while stirring. Succinic anhydride and triethylamine (Et 3 N) were added to the stirring solution of resveratrol to produce crude resveratrol tri-valinate-hemisuccinate. The crude resveratrol tri- valinate-hemisuccinate was loaded (in DCM) onto a silica gel column packed in hexanes.
  • DCM dichloromethane
  • Et 3 N triethylamine
  • the product was brought down in step gradient beginning with 30% ethyl acetate (EtOAc)/70% hexane, slowly increasing to 100% ethyl acetate, then adding 5% acetonitrile/95% ethyl acetate and increasing to 80% acetonitrile/20% ethyl acetate. 0.1% trifluoroacetic acid was added in the solvent system for the entire separation. Fractions were collected and tested by thin layer chromatography for purity. Similar fractions containing pure product were combined.
  • EtOAc ethyl acetate
  • Example 8 Resveratrol tri-phenylalaninate synthesis
  • Resveratrol and 4-dimethylaminopyridine were dissolved in tetrahydrofuran (THF) while stirring.
  • boc-phenylalanine and dicyclohexylcarbodiimide were dissolved in tetrahydrofuran while stirring.
  • the resveratrol solution and the phenylalanine solution were combined while stirring.
  • the reaction formed a precipitate containing crude resveratrol tri-phenylalaninate- boc, which was vacuum filtered.
  • the filtered product was loaded in dichloromethane onto a silica gel column packed with hexane.
  • the first sample was used to determine solubility.
  • the sample was mixed with 1 mL of pH 7.4 phosphate buffered saline (PBS) buffer solution to produce a 4 mg/mL solution.
  • PBS phosphate buffered saline
  • the solution was vortexed and filtered to remove undissolved particles.
  • the filtered solution was then diluted in pH 7.4 buffer in triplicate and stored in a refrigerator to reduce hydrolysis.
  • the solutions were allowed to sit at room temperature for 5 minutes and then were analyzed by HPLC.
  • Calibrators were made based on expected solubility. After visually inspecting solubility in the buffer, the calibration curve was determined in order to include points above and below the expected concentration. The calibrators were stored in a refrigerator until ready for analysis by HPLC.
  • trihemisuccinate experienced quick hydrolysis and was quantitated without resveratrol.
  • Resveratrol trihemiglutarate was found to have the highest solubility and was approximately 400 times more soluble than resveratrol.
  • Resveratrol trihemisuccinate and resveratrol tri-valinate-hemisuccinate also showed an improved solubility as compared to resveratrol.
  • resveratrol tri-valinate, resveratrol di-valinate and resveratrol tri-phenylalaninate were insoluble at pH 7.4, these compounds were expected to be freely soluble at pH 4.0 or less.
  • Resveratrol is dissolved in THF.
  • Di-re/t-butyl dicarbonate is added to an amino acid under aqueous conditions to protect the amine group.
  • DMAP is added to the resveratrol while stirring at room temperature.
  • Triethylamine and the protected amino acid are added to the stirring solution of resveratrol and allowed to stir overnight.
  • the amine group is de-protected with a strong acid.
  • Thin- layer chromatography is used to verify disappearance of starting material and appearance of a new polar spot. The solvent of the reaction mixture is evaporated. This product is separated by chromatography on silica gel to isolate the desired amino acid ester product.
  • Example 1 Preparation of a composition containing 100 ⁇ resveratrol trihemiglutarate
  • composition for topical administration was prepared by mixing the following ingredients:
  • composition delivered resveratrol to a wound and improved the healing process in a subject.
  • a 150 ⁇ solution of resveratrol trihemiglutarate was prepared. First, 9.975 mg of resveratrol trihemiglutarate powder was accurately weighed. Next, the 9.975 mg of resveratrol trihemiglutarate powder was dissolved in 5 ml_ of a 25%
  • Example 13 In vivo application of various compositions containing resveratrol or resveratrol esters in a rat model
  • Demarcations were made in a gently widening parallel approximately 1 cm subscapular and 2.2 cm in length.
  • the left subscapular incision served as a control and the right subscapular incision served as the treatment site.
  • the subscapular areas were incised with a #15 blade though the skin and subcutaneous panniculus carnosus muscle.
  • the composition mixture corresponding to each group was then instilled into the right subscapular wound and the incision closed with 5-0 nylon interrupted fashion. Additional mixture was applied to the surface of the treatment site incision.
  • Three biopsies were taken of normal skin at the time of incision and prior to any mixture instillation. Each site was then monitored and photographed daily. Wound gross morphology was noted daily. Sutures were removed and biopsies were taken at day 8 of the study (postoperative day #7) and sent to pathology for histology review. Histology was reviewed independently by two dermatopathologists.
  • the Compositions of Study Groups 1 -6 were: (1 ) Ca ++ /Mg ++ /siRNA (MCP-1 inhibitor)/hyaluronic acid tetramer in 8% hydroxypropyl methyl cellulose gel; (2) 100 ⁇ resveratrol trihemiglutarate in 8% hydroxypropyl methyl cellulose gel; (3) 400 ⁇ resveratrol trihemiglutarate in 8% hydroxypropyl methyl cellulose gel; (4) 100 ⁇ resveratrol/Ca ++ / g ++ /siRNA (MCP-1 inhibitor)/hyaluronic acid tetramer in 8% hydroxypropyl methyl cellulose gel; (5) 100 ⁇ resveratrol
  • MCP-1 inhibitor trihemiglutarate/Ca ++ /Mg ++ /siRNA (MCP-1 inhibitor)/hyaluronic acid tetramer in 8% hydroxypropyl methyl cellulose gel.
  • siRNA siRNA
  • FIG. 5A is a microscopic image of an untreated control incision.
  • FIG. 5B is a microscopic image of an incision treated with a resveratrol ester. The treated incision showed notably uniform epidermal repair as compared to the deep indention of the epidermis seen in the untreated control incision. The differences in fibrosis and mononuclear dermal infiltrates were not considered significant. Moderate to severe trichogranuloma formation was noted in the specimens.
  • Study Groups 1-5 resulting in 3 animals per study group.
  • An incision, 2 cm in length, will be made on both the right and left shoulder of each rat: the left side will be an untreated control, while the right side will be treated with the Compositions 1-5, with the Study Group number corresponding to the Composition number.
  • compositions 1 -5 will be: (1) 0.5 g resveratrol trihemiglutarate in 1.0 cc
  • aqueous hydroxypropyl methyl cellulose gel (resveratrol trihemiglutarate
  • composition will be applied to the right incision just prior to closure using interrupted 5-0 nylon sutures.
  • the left incision will also be closed using interrupted 5-0 nylon sutures.
  • Each incision will be photographed and measurements will be taken, each day for 7 days.
  • serum blood samples will be taken for systemic absorption assay.
  • a punch biopsy will be taken from each test and control incision.
  • MMP-9 Mestrix metalloproteinase 9
  • TGF- ⁇ Transforming growth factor-beta 1
  • HA hyaluronan
  • EGFR epidermal growth factor receptor
  • CD44 co-localization in lipid rafts
  • Busch, F. et al. "SIRT-1 is required for the inhibition of apoptosis and inflammatory responses in human tenocytes", Journal of Biological Chemistry, Vol. 287, Issue 31 , pp. 25770-25781 (July 27, 2012).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne un ester de resvératrol qui a la structure suivante : (I). R1, R2 et R3 représentent H ou (II). Chaque R4 représente indépendamment une chaîne carbonée de 2 à 4 atomes de carbone comprenant une partie acide carboxylique terminale, une chaîne carbonée de 1 à 5 atomes de carbone comprenant une partie amine ou (III). R5 représente une chaîne carbonée de 3 ou 4 atomes de carbone ayant une partie acide carboxylique terminale. Au moins l'un parmi R1, R2 et R3 représente (IV). L'invention concerne également des sels d'esters de resvératrol.
PCT/US2017/020980 2017-03-06 2017-03-06 Esters de resvératrol WO2018164662A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2020081654A1 (fr) * 2018-10-16 2020-04-23 President And Fellows Of Harvard College Composés d'activation de sirt1

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020081654A1 (fr) * 2018-10-16 2020-04-23 President And Fellows Of Harvard College Composés d'activation de sirt1
US12252505B2 (en) 2018-10-16 2025-03-18 President And Fellows Of Harvard College SIRT1 activating compounds

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