[go: up one dir, main page]

WO2025175304A2 - Process for the preparation of deoxycholic acid - Google Patents

Process for the preparation of deoxycholic acid

Info

Publication number
WO2025175304A2
WO2025175304A2 PCT/US2025/016340 US2025016340W WO2025175304A2 WO 2025175304 A2 WO2025175304 A2 WO 2025175304A2 US 2025016340 W US2025016340 W US 2025016340W WO 2025175304 A2 WO2025175304 A2 WO 2025175304A2
Authority
WO
WIPO (PCT)
Prior art keywords
formula
compound
deoxycholic acid
pharmaceutically acceptable
acceptable salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/016340
Other languages
French (fr)
Other versions
WO2025175304A3 (en
Inventor
Hubert Botte
Jean-Pierre Demoute
Bruno DUDOT
Christelle GEOFFROY
Loïc PAQUIN
Artur PINTO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sanofi Pasteur Inc
Original Assignee
Sanofi Pasteur Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sanofi Pasteur Inc filed Critical Sanofi Pasteur Inc
Publication of WO2025175304A2 publication Critical patent/WO2025175304A2/en
Publication of WO2025175304A3 publication Critical patent/WO2025175304A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C07J1/0003—Androstane derivatives
    • C07J1/0011—Androstane derivatives substituted in position 17 by a keto group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J13/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having a carbon-to-carbon double bond from or to position 17
    • C07J13/007—Normal steroids containing carbon, hydrogen, halogen or oxygen having a carbon-to-carbon double bond from or to position 17 with double bond in position 17 (20)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use

Definitions

  • Outer membrane vesicles are nanosized spherical proteoliposomes that are derived from the outer membrane of Gram-negative bacteria.
  • OMVs or dOMVs have received increased attention as protein or antigen carriers in vaccines against bacterial pathogens (Frontiers in Immunology, 2014, 5, Article 121 , 1-6).
  • the present disclosure provides a process for preparing a compound of formula (7), comprising the step of reacting a compound of formula (6) with a reducing agent:
  • Fig. 1 Protein content in dOMV extracted by animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC of the present disclosure determined by Lowry Peterson assay.
  • Fig. 2 SDS-Page of dOMV extracts (5 and 10 pg).
  • C1 animal derived NaDOC extraction
  • C2 synthetic NaDOC extraction.
  • Fig. 4 Protein content in dOMV extracted by animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC of the present disclosure determined by Lowry Peterson assay at different steps of the manufacturing process of the Drug Substance (DS): from the post benzonase treatment (post OMV extraction), through the recovery step and the purification steps, to the final DS composition.
  • NaDOC animal derived Sodium Deoxycholate
  • DS Drug Substance
  • Each and every hydrogen in the compound includes all isotopes of hydrogen, in particular 1 H and 2 H (deuterium).
  • human or animal-derived substance refers to a substance, such as a protein, a lipid, a steroid, a monosaccharide or a polysaccharide, originating from a human or non-human animal, that is obtained from, e.g. extracted from, a human or non-human animal.
  • the source of hydride may be sodium borohydride with PdCh diisobutylaluminium hydride, lithium tri-tert- butoxyaluminium hydride (LTBA), lithium tri-sec-butylborohydride (L-Selectride) or lithium aluminium hydride.
  • the reducing agent is lithium tri-tert-butoxyaluminium hydride or lithium aluminium hydride.
  • the reaction solvent may be THF.
  • the source of hydride is lithium tri-tert-butoxyaluminium hydride
  • the reaction solvent is THF
  • the reaction is conducted at a temperature of between -20 and 20°C, in particular at a temperature of between -5 and 5°C, such as at 0°C.
  • the use of lithium aluminium hydride is also particularly advantageous in an industrial setting owing to its low molecular weight, which allows a comparative reduction in the volume of reducing agent required.
  • the source of hydride is lithium aluminium hydride and the reaction solvent is THF.
  • the source of hydride is lithium aluminium hydride
  • the reaction solvent is THF
  • the reaction is conducted at a temperature of between -20 and 20°C, in particular at a temperature of between -15 and 15°C, such as at 10°C.
  • PG is a silyl ether protecting group, for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tertbutyldimethylsilyl (TBS) ether, or tert-butyldiphenylsilyl (TBDPS) ether.
  • TMS trimethylsilyl
  • TES triethylsilyl
  • TIPS triisopropylsilyl
  • DEIPS diethylisopropylsilyl
  • TBS tertbutyldimethylsilyl
  • TDPS tert-butyldiphenylsilyl
  • PG is tertbutyldimethylsilyl (TBS) ether.
  • PG is a silyl ether protecting group, for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert-butyldimethylsilyl (TBS) ether, tert-butyldipheny Isily I (TBDPS) ether, and the source of hydride is sodium borohydride with PdCh, diisobutylaluminium hydride, lithium tri-tert-butoxyaluminium hydride (LTBA), lithium tri-sec- butylborohydride (L-Selectride) or lithium aluminium hydride.
  • TMS trimethylsilyl
  • TES triethylsilyl
  • TIPS triisopropylsilyl
  • DEIPS diethylisopropylsilyl
  • TBS tert-but
  • the process further comprises the step of modifying the compound of formula (7) to form a compound of formula (9):
  • the compound of formula (9) may be prepared by reacting the compound of formula (8) with chloromethyl methyl ether (MOM), 3,4-dihydropyran (THP), isobutylene ( f Bu), trimethylsilyl chloride (TMS), triethylsilyl chloride (TES), Ci-e alkyl halide (Ci-e alkyl ester), acetyl chloride (Ac), pivaloyl chloride (Piv), benzoyl chloride (Bz), picolinoyl chloride (Pico), chloromethyl methyl carbonate, chloromethyl ethyl carbonate, di-tert-butyl carbonate (Boc) or N-phenyl carbamoyl chloride in the presence of a base.
  • MOM chloromethyl methyl ether
  • THP 3,4-dihydropyran
  • f Bu isobutylene
  • TMS trimethylsilyl chloride
  • TES triethylsilyl chlor
  • the PG for the compounds of formula (6) and (7) is a silyl ether protecting group and the PGs for the compound of formula (9) are both ester protecting groups.
  • the deprotection reaction is suitable to remove the protecting groups and hydrolyse the -CO2R 4 group to a CO2H group in a single step using one or more reagents in one-pot, wherein the PGs and R 4 are as defined hereinbefore for the compounds of formula (9), (10) and (1 1).
  • the compound of formula (6) is prepared by reacting a compound of formula (5) with an oxidising agent:
  • the compound of formula (6) is prepared by reacting the compound of formula (5) with an oxidising agent, for example with a mixture of (COCI)2 or TFAA (trifluoroacetic anhydride), DMSO and triethylamine.
  • an oxidising agent for example with a mixture of (COCI)2 or TFAA (trifluoroacetic anhydride), DMSO and triethylamine.
  • the compound of formula (6) is prepared by reacting the compound of formula (5) with an oxidising agent, for example with a mixture of TFAA, DMSO and triethylamine, wherein the reaction is conducted at between -20°C to -10°C, such as -15°C.
  • the reaction solvent is dichloromethane.
  • the use of the Swern oxidation employing TFAA as the DMSO-activating agent was found to be particularly advantageous, because in addition to not requiring the utilisation of expensive or toxic reagents, it was found that the reaction proceeded cleanly and effectively at -20°C to -10°C, such as -15°C.
  • the compound of formula (5) is prepared by reacting a compound of formula (4) with an ethyltriphenylphosphonium salt: wherein PG is as defined hereinbefore for the compounds of formula (5), (6) and (7).
  • the compound of formula (5) is prepared by reacting the compound of formula (4) with an ethyltriphenylphosphonium salt, such as ethyltriphenylphosphonium bromide or iodide in the presence of a base, such as sodium bis(trimethylsilyl)amide (NaHMDS), lithium diisopropylamide (LDA), butyllithium (BuLi) or potassium tert-butoxide (KO‘Bu).
  • a base such as sodium bis(trimethylsilyl)amide (NaHMDS), lithium diisopropylamide (LDA), butyllithium (BuLi) or potassium tert-butoxide (KO‘Bu).
  • the ethyltriphenylphosphonium salt is ethyltriphenylphosphonium bromide
  • the base is potassium tert-butoxide.
  • the solvent may be THF, methyl-THF, dioxane, diethyl ether, dimethoxyethane, or methyl tert-butyl ether.
  • the compound of formula (4) is prepared by reacting a compound of formula (3) with PG-X: wherein PG is as defined hereinbefore for the compounds of formula (4), (5), (6) and (7), and X is a leaving group.
  • the oxidising agent is molecular oxygen (O2)
  • the source of copper selected from Cu(MeCN)4PFs and Cu(0tf)2.
  • the reaction may also proceed in the presence of a reductant such as benzoin with triethylamine, FeBr2, Zn powder, EtsSiH or sodium ascorbate.
  • the solvent is acetone or a 1 :1 mixture of acetone and methanol.
  • the amino pyridine derivative of formula (a2) is commercially available or may be obtained from commercially available starting materials and prepared from literature procedures.
  • the compound of formula (2) is prepared by reacting a compound of formula (1) with an amino pyridine derivative of formula (a), such as (a1) or (a2) in the presence of p-toluenesulfonic acid monohydrate or molecular sieves and a toluene solvent, and heating the reaction mixture to reflux in a Dean-Stark apparatus.
  • a compound of formula (2a) is prepared by reacting the compound of formula (1) with an amino pyridine derivative of formula (a2):
  • the compound of formula (1) is known as etiocholanolone. It is commercially available from chemical suppliers. The use of etiocholanolone in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof is particularly beneficial because the stereochemistry required at the C 3 and C 5 of deoxycholic acid is already contained within etiocholanolone:
  • this embodiment provides a comparatively simplified synthesis because the number of synthetic steps and/or comparative complexity of the chemistry required to access deoxycholic acid from other scaffolds (wherein the stereochemistry required at the C 3 and C 5 of deoxycholic acid is not contained) are reduced.
  • This process advantageously combines the beneficial effects described hereinbefore associated with the Wittig reaction, the oxidation reaction, and the reduction reaction.
  • the present disclosure also provides a process for preparing a compound of formula (8), comprising the step of reacting a compound of formula (6) with a reducing agent to provide a compound of formula (7):
  • the present disclosure also provides a process for preparing a compound of formula (8), comprising the step of deprotecting a compound of formula (7) to provide a compound of formula (8):
  • the present disclosure provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, comprising the step of deprotecting a compound of formula (7a) to provide a compound of formula (8).
  • the present disclosure also provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, wherein the process comprises any of the process steps or processes described herein.
  • the present disclosure also provides deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes defined hereinbefore.
  • Reagent to precipitate a component of a composition for instance an unconjugated free polysaccharide from carrier protein-bound material (see for instance Q.P. Lei et al., Developments in Biologicals 2000, 103, 259-264).
  • the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable according to any one of the processes described herein in the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition.
  • the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable according to any one of the processes described herein in the manufacturing process of a pharmaceutical or immunogenic composition.
  • the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable according to any one of the processes described herein in the manufacturing process of an immunogenic composition.
  • the immunogenic composition may be a vaccine.
  • the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial.
  • the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance.
  • the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to any of the processes described herein for extracting outer membrane vesicles from Gram-negative bacteria.
  • the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein for extracting outer membrane vesicles from Gram-negative bacteria.
  • animal-derived bile acid products may contain animal pathogens and other harmful agents such as animal or microbial metabolites and toxins, including bacterial toxins such as pyrogens. Consequently, such animal-derived products must be subjected to expensive purification processes.
  • One embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for extracting outer membrane vesicles from Gram-negative bacteria, in particular from Neisseria meningitidis.
  • One embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for use in extracting outer membrane vesicles from Gram-negative bacteria, in particular from Neisseria meningitidis serogroup B (MenB).
  • One further embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for use in extracting outer membrane vesicles from Gram-negative bacteria, in particular from Neisseria meningitidis serogroup B (MenB).
  • One further embodiment provides a composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein and an outer membrane vesicle (OMV).
  • Such compositions may be pharmaceutical or immunogenic compositions.
  • the pharmaceutical or immunogenic composition typically further comprises one or more pharmaceutically acceptable excipients.
  • the immunogenic composition may be a vaccine.
  • the extraction process may be free of any human or animal-derived substance.
  • One embodiment relates to the use of a compound of formula (1), (2), (2a), (3), (4), (4a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10), (10a), (11) or (11 a) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof.
  • Another embodiment relates to a process for manufacturing a pharmaceutical, immunogenic or cosmetic composition comprising at least one step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein.
  • the present disclosure provides a manufacturing process of a pharmaceutical or immunogenic composition comprising at least a step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein.
  • the present disclosure provides a manufacturing process of an immunogenic composition comprising at least a step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein.
  • the immunogenic composition may be a vaccine.
  • the extraction process may be free of any human or animal-derived substance.
  • the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial and/or the deoxycholic acid or a pharmaceutically acceptable salt is prepared/obtainable by any of the processes described herein.
  • the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be a component of the final pharmaceutical, immunogenic or cosmetic composition but such manufacturing processes of a pharmaceutical, immunogenic or cosmetic composition using the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof are not a production process of the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof.
  • the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. Therefore, in one embodiment, the present disclosure provides the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance, as a detergent for the extraction of complexes comprising lipid(s) and protein(s) from bacteria, for instance proteoliposomes, proteasomes, proteolipidic vesicles, etc.
  • Complexes comprising lipid(s) and protein(s) are complexes that comprise at least one protein and at least one lipid and that may comprise more than one lipid and/or protein.
  • the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial and/or the deoxycholic acid or a pharmaceutically acceptable salt is prepared/obtainable by any of the processes described herein.
  • the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein.
  • a treatment with a nuclease, like benzonase, according to any methods known in the art can be used to digest nucleic acids after formation of the outer membrane vesicles and before recovery of the outer membrane vesicles.
  • the present disclosure also provides a compound of formula (6) or (7) wherein PG is as defined hereinbefore.
  • the present disclosure provides a compound of formula (2), (2a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10), (10a), (11) or (1 1a):
  • the organic phase (1) was then washed with 1 N HCI (2 x 250 mL, 2 x 5V).
  • the acidic aqueous phase (product 3 was detected in this phase) was extracted with EtOAc (3 x 100 mL, 3 x 2V) to provide organic phase (2).
  • the combined organic phases (1) + (2) were then washed with a concentrated aqueous solution of sodium metabisulphite (Na2S20s) (250 g in 500 mL of water), and the presence of peroxide in the organic phase was checked using peroxide test strips.
  • Na2S20s sodium metabisulphite
  • reaction mixture was allowed to stir at ⁇ -60°C for at least 10 minutes, after which point a solution of compound 5a (13 g, 30 mmol) in DCM (30 ml_, 2.3V) was added dropwise thereto at the same temperature. During and following the addition of compound 5a a further exotherm was observed.
  • the reaction mixture was allowed to stir at a temperature ⁇ -60° C for 2 hours, after which point triethylamine (22.5 mL, 162 mmol, 5.4 eq) was added dropwise thereto. During and following the addition of triethylamine a further exotherm was observed.
  • the reaction mixture was stirred and allowed to warm to room temperature over a duration of 16.5 hours.
  • reaction mixture was stirred for an additional hour at -15°C and then allowed to warm to room temperature over a duration of 12 hours. Upon completion, the reaction mixture was poured into 100 mL of water and extracted with diisopropyl ether (3 x 50 mL), the combined organic extracts were then washed with a saturated aqueous solution of ammonium chloride (3 x 40 mL), dried over MgSO4 and filtered. The organic phase was then concentrated in vacuo.
  • reaction mixture Upon completion, the reaction mixture was cooled to 10°C, and then ethyl acetate (20 mL) was added dropwise over 30 minutes. The reaction mixture obtained was then poured onto crushed ice (200 g) and ethyl acetate (100 mL). The resulting mixture was then acidified to pH 3 with aqueous HCI 3N. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (3 x 50 mL). The combined organic phases were dried over MgSCM and filtered, and the filtrate was concentrated in vacuo.
  • reaction mixture was red. Reaction progress was monitored by TLC (heptane/DIPO 80:20, PMA stain).
  • reaction mixture was poured into water (500 mL, 27V). The mixture was then decanted, and the organic phase was separated (yellow oil decanted). The aqueous phase was then extracted with EtOAc (3 x 100 mL, 3 x 5.4V).
  • reaction mixture was filtered through celite, and the celite was rinsed with MeOH (1V) and the MeOH was concentrated to dryness. To remove any remaining insoluble particulate matter, the product was filtered through a silica plug and eluted with a heptane/EtOAc 8:2 mixture to provide a white solid after evaporation (3.6 g).
  • reaction vessel To a reaction vessel were added compound 11 a (3.3 g, 6.7 mmol), water (33 mL, 10V), methanol (100 mL, 30V) and KOH (6.7 g, 1 19.7 mmol, 17.8 eq). The reaction mixture was then heated at reflux for 20 hours. Reaction progress was monitored by NMR, and the reaction was considered complete when the NMR no longer contained signals at 5.14-5.05 and 4.81-4.63 ppm. Upon completion of the reaction, the methanol was evaporated, and water was added to the resulting residue. The aqueous solution was then washed with EtOAc (100 ml_, 30V).
  • the precipitate was drained and rinsed with water.
  • the precipitate was then taken up with 1 eq of 1 N NaOH and heated under reflux with stirring for 20 minutes.
  • the solution was then cooled to 20°C and filtered through celite.
  • the filtrate was then acidified to pH 1 , and the resulting suspension was stirred for 30 minutes.
  • the precipitate was then drained, rinsed with water and dried under vacuum in the presence of P2O5.
  • the deoxycholic acid prepared by the process of Example 1 is a synthetic deoxycholic acid free of any human or animal-derived substance as the process of Example 1 relies on the use of non-animal derived starting materials and reagents.
  • Extraction was performed by mixing 1 .7X extraction buffer (either with animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC (compound (III) from Example 1 i.e. free of any human or animal-derived substance) and recovered biomass to achieve a ratio detergent/biomass of 0.5%.
  • Extraction was performed by mixing 1 ,7X extraction buffer (see table 1 above) (either with animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC (compound (III) from Example 1 , that is, free of any human or animal-derived substance) and recovered biomass to achieve a ratio detergent/biomass of 0.5%.
  • 1 ,7X extraction buffer see table 1 above
  • NaDOC animal derived Sodium Deoxycholate
  • synthetic NaDOC compound (III) from Example 1 , that is, free of any human or animal-derived substance
  • Protein quantification was determined by Lowry assays. Protein profile was assessed by SDS-Page. Capsular polysaccharide content was determined by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).

Landscapes

  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Birds (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dermatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Steroid Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present disclosure relates to a process for preparing a compound of formula (7), comprising the step of reacting a compound of formula (6) with a reducing agent, and to process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof comprising said step, and to uses of the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared by the process to extract outer membrane vesicles from Gram-negative bacteria.

Description

PROCESS FOR THE PREPARATION OF DEOXYCHOLIC ACID
TECHNICAL FIELD
The present disclosure relates to a process for the preparation of a compound of formula (7), and its use in the preparation of deoxycholic acid or a pharmaceutically acceptable salt thereof, and the use of deoxycholic acid prepared by the process for the extraction of outer membrane vesicles.
TECHNICAL BACKGROUND
Deoxycholic acid is a bile acid. As shown by formula (I), it has the following structure:
Deoxycholic acid has numerous applications in both the pharmaceutical and cosmetic industries. For example, deoxycholic acid is used as a detergent for the extraction of outer membrane vesicles (OMVs) (also known as detergent-extracted outer membrane vesicles (dOMVs) following their extraction).
Outer membrane vesicles are nanosized spherical proteoliposomes that are derived from the outer membrane of Gram-negative bacteria. In recent years OMVs or dOMVs have received increased attention as protein or antigen carriers in vaccines against bacterial pathogens (Frontiers in Immunology, 2014, 5, Article 121 , 1-6).
Pharmaceutical grade bile acid preparations containing deoxycholic acid are commercially available at low cost from animal carcasses. However, as with all products from animal sources, there is a risk that animal- derived bile acid products may contain animal pathogens and other harmful agents such as animal or microbial metabolites and toxins, including bacterial toxins such as pyrogens. These harmful agents can be removed, but expensive purification processes are required.
In view of this risk, and the economic disadvantages associated with expensive purification processes, synthetic bile acids such as deoxycholic acid have been prepared from compounds derived from non-animal sources, such as from plant sources or synthetic starting materials (see W02008157635, WO2017211820, WO2019024920, EP3284748 and J. Org Chem, 2007, 72, 9298-9307). But there is still a need for a process to produce synthetic deoxycholic acid according to Pharmaceutical Good Manufacturing Practices (GMP). And there is a need for such a process that is scalable up to industrial scale. Accordingly, the present disclosure describes a particularly useful synthesis of deoxycholic acid starting from compounds derived from non-animal sources.
SUMMARY
In a first aspect, the present disclosure provides a process for preparing a compound of formula (7), comprising the step of reacting a compound of formula (6) with a reducing agent:
(6) (7) wherein PG is a protecting group.
In a second aspect, the present disclosure provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof wherein the process comprises the process as defined by the first aspect. In particular, the deoxycholic acid or its pharmaceutically acceptable salt is a pharmaceutical grade material or a GMP grade material.
In a further aspect there is provided a use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the processes of the present disclosure for the extraction of outer membrane vesicles (OMVs) from Gram-negative bacteria, in particular, outer membrane vesicles from Neisseria meningitidis serogroup B (MenB).
In yet a further aspect there is provided a process for extracting outer membrane vesicles from Gramnegative bacteria using deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the processes of the present disclosure.
In yet a further aspect there is provided a pharmaceutical, immunogenic or cosmetic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the processes of the present disclosure. In a particular aspect there is provided a pharmaceutical or immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the processes of the present disclosure. In a further particular aspect there is provided an immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the processes of the present disclosure. The immunogenic composition may be a vaccine.
The present disclosure will now be described with reference to the accompanying drawings, in which: Fig. 1 Protein content in dOMV extracted by animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC of the present disclosure determined by Lowry Peterson assay.
Fig. 2 SDS-Page of dOMV extracts (5 and 10 pg). C1 : animal derived NaDOC extraction; C2: synthetic NaDOC extraction.
Fig. 3 Capsular polysaccharide content determined by HPSEC
Fig. 4 Protein content in dOMV extracted by animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC of the present disclosure determined by Lowry Peterson assay at different steps of the manufacturing process of the Drug Substance (DS): from the post benzonase treatment (post OMV extraction), through the recovery step and the purification steps, to the final DS composition. DEFINITIONS
The term ‘optionally substituted’ as used herein refers to a group which may be unsubstituted or substituted by a substituent as herein defined.
The prefix “Cx-y” (where x and y are integers) as used herein refers to the number of carbon atoms in a given group. Thus, a C1-6 alkyl group contains from 1 to 6 carbon atoms.
Each and every hydrogen in the compound (such as in an alkyl group or where referred to as hydrogen) includes all isotopes of hydrogen, in particular 1H and 2H (deuterium).
The term ‘C1-6 alkyl’ as used herein as a group or part of a group refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms respectively. Examples of such groups include methyl, ethyl, n-propyl, pentyl, hexyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like. The term “aryl” as used herein refers to a carbon-containing aromatic group, such as phenyl.
The terms “synthetic deoxycholic acid” as used herein refers to deoxycholic acid or pharmaceutically acceptable salt thereof made by chemical synthesis in contrast to the natural deoxycholic acid which is extracted from animals. A synthetic deoxycholic acid “free of any human or animal-derived substance” refers to synthetic deoxycholic acid or pharmaceutically acceptable salt thereof made by chemical synthesis which does not use any human or animal-derived substance, e.g. as starting materials or reagents. Within the scope of the present disclosure, the expression “human or animal-derived substance” refers to a substance, such as a protein, a lipid, a steroid, a monosaccharide or a polysaccharide, originating from a human or non-human animal, that is obtained from, e.g. extracted from, a human or non-human animal.
The terms “pharmaceutically acceptable salt” as used herein refers to any salt of deoxycholic acid that is pharmaceutically acceptable, like sodium salt of deoxycholic acid (sodium deoxycholate). Further examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts.
DETAILED DESCRIPTION
Process for preparing deoxycholic acid or pharmaceutically acceptable salt thereof
The present disclosure provides a process for preparing deoxycholic acid or pharmaceutically acceptable salt thereof comprising any one of or any combination of the process steps described herein. Accordingly, the present disclosure provides a process for preparing deoxycholic acid or pharmaceutically acceptable salt thereof comprising the process for preparing the compound of formula (2) or (2a) from the compound of formula (1), and/or the process for preparing the compound of formula (3) from the compound of formula (2) or (2a), and/or the process for preparing the compound of formula (4) or (4a) from the compound of formula (3), and/or the process for preparing the compound of formula (5) from the compound of formula
(4) or (4a), and/or the process for preparing the compound of formula (6) or (6a) from the compound of formula (5), and/or the process for preparing the compound of formula (7) or (7a) from the compound of formula (6) or (6a) respectively, and/or the process for preparing the compound of formula (8) from the compound of formula (7) or (7a), and/or the process for preparing the compound of formula (9) or (9a) from the compound of formula (8), and/or the process for preparing the compound of formula (10) or (10a) from the compound of formula (9) or (9a) respectively, and/or the process for preparing the compound of formula (11) or (11a) from the compound of formula (10) or (10a) respectively, and/or the process for preparing the compound of formula (I) from the compound of formula (11) or (11 a), and/or the process for preparing the compound of formula (II) or (III) from the compound of formula (I), as described herein.
In particular, the present disclosure provides a process for preparing deoxycholic acid or pharmaceutically acceptable salt thereof comprising the process for preparing the compound of formula (2) or (2a) from the compound of formula (1), and/or the process for preparing the compound of formula (6) or (6a) from the compound of formula (5), and/orthe process for preparing the compound of formula (7) from the compound of formula (6) or (6a), as described herein. In particular, the present disclosure provides a process for preparing deoxycholic acid or pharmaceutically acceptable salt thereof comprising the process for preparing the compound of formula (2) or (2a) from the compound of formula (1), the process for preparing the compound of formula (6) or (6a) from the compound of formula (5), and the process for preparing the compound of formula (7) or (7a) from the compound of formula (6) or (6a) respectively, as described herein. The present disclosure also provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof comprising the process for preparing the compound of formula (7) as described hereinbelow. Process for preparing the compound of formula (7)
The present disclosure provides a process for preparing the compound of formula (7), comprising the step of reacting a compound of formula (6) with a reducing agent:
(6) (7) wherein PG is a protecting group.
The reduction reaction is diastereoselective, with a diastereomeric ratio (a/p) of at least 60/40, 70/30, 80/20, 85/15, 90/10 or 95/5 in favour of the a-isomer of the compound formula (7):
In particular, the reduction reaction is diastereoselective with a diastereomeric ratio (a/p) of at least 85/15 or 90/10 or 95/5 in favour of the a-isomer of the compound of formula (7).
The reducing agent typically contains a source of hydride (H~) for example a metal hydride or metal borohydride reagent.
The source of hydride may be sodium borohydride with PdCh diisobutylaluminium hydride, lithium tri-tert- butoxyaluminium hydride (LTBA), lithium tri-sec-butylborohydride (L-Selectride) or lithium aluminium hydride. In particular, the reducing agent is lithium tri-tert-butoxyaluminium hydride or lithium aluminium hydride.
The reaction solvent may be THF.
It was found that the use of lithium tri-tert-butoxyaluminium hydride as the reducing agent provided beneficial diastereoselectivity towards the a-isomer of the compound of formula (7) (12a/12p = 92/8). This advantageously provides the stereochemistry required for the alcohol functionality at the C12 position of the deoxycholic acid scaffold and minimises the degree of purification required to remove the unwanted p- isomer.
In one embodiment, the source of hydride is lithium tri-tert-butoxyaluminium hydride, the reaction solvent is THF, and the reaction is conducted at a temperature of between -20 and 20°C, in particular at a temperature of between -5 and 5°C, such as at 0°C.
It was also found that the use of lithium aluminium hydride as the reducing agent provided beneficial diastereoselectivity towards the a-isomer of the compound of formula (7) (12a/12p = 97/3). This advantageously provides the stereochemistry required for the alcohol functionality at the C12 position of the deoxycholic acid scaffold and minimises the degree of purification required to remove the unwanted p- isomer. The use of lithium aluminium hydride is also particularly advantageous in an industrial setting owing to its low molecular weight, which allows a comparative reduction in the volume of reducing agent required.
In one embodiment, the source of hydride is lithium aluminium hydride and the reaction solvent is THF.
In one embodiment, the source of hydride is lithium aluminium hydride, the reaction solvent is THF, and the reaction is conducted at a temperature of between -20 and 20°C, in particular at a temperature of between -15 and 15°C, such as at 10°C.
In one embodiment, for the compound of formula (6) and (7), PG is selected from an ether, silyl ether, ester, carbonate or carbamate protecting group.
For example, PG may be selected from methoxymethyl (MOM) ether, benzyloxymethyl (BOM) ether, tetrahydropyranyl (THP) ether, tert-butyl ether fBu), allyl ether, para-methoxyphenyl (PMP) ether, benzyl ether (Bn), para-methoxybenzyl (PMB) ether, triphenylmethyl ether (Trit), -SiRs ether wherein R = C1-6 alkyl and/or aryl, trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert-buty Idimethy Isily I (TBS) ether, tert-buty Idipheny Isi lyl (TBDPS) ether, C1-6 alkyl ester, acetate ester (Ac), pivaloate ester (Piv), benzoate ester (Bz), picolinate ester (Pico), methyl carbonate, ethyl carbonate, tert-butyl carbonate (Boc), allyl carbonate (Alloc), benzyl carbonate or N-phenyl carbamate.
In a particular embodiment, PG is a silyl ether protecting group, for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tertbutyldimethylsilyl (TBS) ether, or tert-butyldiphenylsilyl (TBDPS) ether. In particular, PG is tertbutyldimethylsilyl (TBS) ether. Methods of protecting functional groups, can be found in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999). In one embodiment, PG is a silyl ether protecting group, for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert-butyldimethylsilyl (TBS) ether, tert-butyldipheny Isily I (TBDPS) ether, and the source of hydride is sodium borohydride with PdCh, diisobutylaluminium hydride, lithium tri-tert-butoxyaluminium hydride (LTBA), lithium tri-sec- butylborohydride (L-Selectride) or lithium aluminium hydride. In a particular embodiment the PG is tert- butyldimethylsilyl (TBS) ether and the reducing agent is lithium tri-tert-butoxyaluminium hydride.
In a further embodiment, the process further comprises the step of modifying the compound of formula (7) to form a compound of formula (9):
(7) (9) wherein the PGs are independently as defined hereinbefore for the compounds of formula (6) and (7).
In a further embodiment, the process further comprises the step of deprotecting the compound of formula (7) to provide a compound of formula (8):
(7) (8) and protecting the alcohol groups by reacting the compound of formula (8) with PG-X to form a compound of formula (9): wherein the PGs are independently as defined hereinbefore for the compound of formula (7) and X is a leaving group.
In this embodiment, for the compound of formula (7), the PG may be a silyl ether protecting group, for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert-butyldimethylsilyl (TBS) ether, or tert-butyldiphenylsilyl (TBDPS) ether.
Methods of deprotecting functional groups, can be found in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999). In a particular embodiment, PG is tert-butyldimethylsilyl (TBS) ether, and the deprotection reaction is suitable to remove the TBS group to provide the compound of formula (8). For example, the deprotection reaction requires treating the compound of formula (7) with tetra-n-butylammonium fluoride (TBAF) in an appropriate solvent, such as tetrahydrofuran (THF).
For the protection step, where the alcohol groups of the compound of formula (8) are protected with PGs following a reaction with PG-Xto form a compound of formula (9), the PGs are independently selected from ether, silyl ether, ester, carbonate or carbamate protecting groups, and X is a leaving group.
For example, the PGs are independently selected from methoxymethyl (MOM) ether, tetrahydropyranyl (THP) ether, tert-butyl ether fBu), trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, C1-6 alkyl ester, acetate ester (Ac), pivaloate ester (Piv), benzoate ester (Bz), picolinate ester (Pico), methyl carbonate, ethyl carbonate, tert-butyl carbonate (Boc), or N-phenyl carbamate.
For example, the compound of formula (9) may be prepared by reacting the compound of formula (8) with chloromethyl methyl ether (MOM), 3,4-dihydropyran (THP), isobutylene (fBu), trimethylsilyl chloride (TMS), triethylsilyl chloride (TES), Ci-e alkyl halide (Ci-e alkyl ester), acetyl chloride (Ac), pivaloyl chloride (Piv), benzoyl chloride (Bz), picolinoyl chloride (Pico), chloromethyl methyl carbonate, chloromethyl ethyl carbonate, di-tert-butyl carbonate (Boc) or N-phenyl carbamoyl chloride in the presence of a base.
In one embodiment, for the compound of formula (9), the PGs are both ester protecting groups, for example a C1-6 alkyl ester protecting group, acetate ester, pivaloate ester, benzoate ester or picolinate ester group. In particular, the PGs are acetate ester protecting groups, that is PG-0 are both Ac-0 wherein Ac is an acetyl group (CH3CO-).
When the PGs are both acetyl, the compound of formula (9) may be prepared by reacting the compound of formula (8) with an acetyl source such as acetyl chloride or acetic anhydride, in the presence of an organic base such as triethylamine, optionally in the presence of an esterification catalyst, such as 4- dimethylaminopyridine (DMAP) at a temperature of between 15 and 30°C.
The PGs for the compounds of formula (6), (7) and (9) may be independently selected from ether, silyl ether, ester, carbonate or carbamate protecting groups as defined hereinbefore.
In one embodiment, at least the PG for the compounds of formula (6) and (7) differs from the PGs for the compound of formula (9).
In one embodiment the PG for the compounds of formula (6) and (7) is a silyl ether protecting group and the PGs for the compound of formula (9) are both ester protecting groups.
In one embodiment the PG for the compounds of formula (6) and (7) is a silyl ether protecting group for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert-butyldimethylsily I (TBS) ether, or tert-buty Idiphenylsily I (TBDPS) ether; and the PGs for the compound of formula (9) are both ester protecting groups, for example a C1-6 alkyl ester protecting group, acetate ester, pivaloate ester, benzoate ester or picolinate ester group. In particular, the PGs for the compound of formula (9) are acetate ester protecting groups.
In a particular embodiment, the PG for the compounds of formula (6) and (7) is a tert-buty Idimethy Isily I (TBS) ether protecting group, and the PGs for the compound of formula (9) are both acetate ester protecting groups.
In a further embodiment, the process further comprises the step of reacting the compound of formula (9) with a compound of formula (b) in the presence of a coupling agent to form a compound of formula (10): wherein, R4 is C1-6 alkyl, and wherein the PGs are as defined hereinbefore for the compound of formula (9). In one embodiment, R4 is methyl.
The transformation from compound (9) to compound (10) is an example of an “ene” or “Alder-ene” reaction. It is a reaction between an alkene with an allylic hydrogen (the ene) and a compound containing a multiple bond (the enophile), to form a new o-bond with migration of the ene double bond and a 1 ,5-hydrogen shift. The product is a substituted alkene with the double bond shifted to the allylic position. Further details may be found in Strategic Applications of Named Reactions in Organic Synthesis (L. Kurti and B. Czako, 1st Edition; Elsevier, 2005).
The reaction typically takes place in the presence of a coupling agent, for example a Lewis acid, under anhydrous conditions. In one embodiment, the compound of formula (9) is reacted with the compound of formula (b) to form the compound of formula (10), in the presence of a Lewis acid. The reaction may take place in the presence of a Lewis acid that is an organometallic halide, such as methylaluminium dichloride (MeAICL), dimethylaluminium chloride (Me2AICI), ethylaluminium dichloride (EtAICL) and diethylaluminium dichloride (Et2AICl2). In particular, the reaction takes place in the presence of a Lewis acid that is ethylaluminium dichloride (EtAICh).
In one embodiment, the compound of formula (9) is reacted with the compound of formula (b) wherein R4 is methyl (i.e. methyl propiolate) to form the compound of formula (10), in the presence of a Lewis acid, in particular ethylaluminium dichloride (EtAICL), at a temperature of between 15 and 30°C.
The compounds of formula (b) are commercially available or may be obtained from commercially available starting materials, prepared from literature procedures.
In a further embodiment, the process further comprises the step of reducing the compound of formula (10) to form a compound of formula (11) using a reducing agent: wherein the PGs and R4 are as defined hereinbefore for the compounds of formula (9) and (10). In one embodiment, the reducing agent is H2. In one embodiment, the reducing step is performed with H2 under hydrogenation conditions, such as an atmosphere of H2 in the presence of a transition metal catalyst, for example Pd/C (palladium on carbon).
In one embodiment, the step of reducing the compound of formula (10) to form the compound of formula (11) is performed with H2 in the presence of Pd/C, in an appropriate hydrogenation solvent such as an alcohol or THF. In particular, the reaction solvent is MeOH. Compound (10) may be subjected to H2 delivered from a balloon, or alternatively the hydrogenation reaction can be performed on a Parr hydrogenator or a Thales H-cube flow reactor.
In one embodiment, the step of reducing the compound of formula (10) to form the compound of formula (11) is performed with H2 in the presence of Pd/C in methanol, at a H2 pressure of 5 bar.
In a further embodiment, the process further comprises the step of deprotecting the protecting groups and hydrolysing the -OR4 group to provide deoxycholic acid (formula I): wherein the PGs and R4 are as defined hereinbefore for the compounds of formula (9), (10) and (1 1).
As described hereinbefore for the compounds of formula (9) and (10), for the compound of formula (11) the PGs are selected from an ether, silyl ether, ester, carbonate or carbamate protecting group, and R4 is C1-6 alkyl.
For example, the PGs are independently selected from methoxymethyl (MOM) ether, tetrahydropyranyl (THP) ether, tert-butyl ether (*Bu), trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, C1-6 alkyl ester, acetate ester (Ac), pivaloate ester (Piv), benzoate ester (Bz), picolinate ester (Pico), methyl carbonate, ethyl carbonate, tert-butyl carbonate (Boc) or N-phenyl carbamate, and R4 is C1-6 alkyl.
In a particular embodiment, the PGs are both ester protecting groups, for example a C1-6 alkyl ester protecting group, acetate ester, pivaloate ester, benzoate ester or picolinate ester group. In particular, the PGs are both acetate ester protecting groups and R4 is methyl.
As described hereinbefore, methods of deprotecting functional groups, can be found in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999) which includes explicit guidance as to the conditions needed to remove a C1-6 alkyl ester, acetate ester, pivaloate ester, benzoate ester and picolinate ester group protecting groups to reveal secondary alcohols, and the conditions needed to hydrolyse the -CO2R4 group (R4 is C1-6 alkyl, in particular methyl) to a CO2H group.
In one embodiment, the process comprises the step of deprotecting the protecting groups and hydrolysing the -OR4 group to provide deoxycholic acid, wherein the PGs are independently selected from an ether, silyl ether, ester, carbonate or carbamate protecting group, and R4 is C1-6 alkyl, in particular wherein the PGs are independently selected from methoxymethyl (MOM) ether, tetra hydro pyranyl (THP) ether, tertbutyl ether (‘Bu), trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, C1-6 alkyl ester, acetate ester (Ac), pivaloate ester (Piv), benzoate ester (Bz), picolinate ester (Pico), methyl carbonate, ethyl carbonate, tert- butyl carbonate (Boc), or N-phenyl carbamate, and R4 is C1-6 alkyl, in particular wherein the PGs are both ester protecting groups, for example a C1-6 alkyl ester protecting group, acetate ester, pivaloate ester, benzoate ester or picolinate ester group.
In one embodiment, the deprotection reaction is suitable to remove the protecting groups and hydrolyse the -CO2R4 group to a CO2H group in a single step using one or more reagents, or in multiple steps using one or more reagents.
In a particular embodiment, the deprotection reaction is suitable to remove the protecting groups and hydrolyse the -CO2R4 group to a CO2H group in a single step using one or more reagents in one-pot, wherein the PGs and R4 are as defined hereinbefore for the compounds of formula (9), (10) and (1 1).
In a particular embodiment, the protecting groups are both acetate esters and R4 is methyl, and the deprotection reaction is suitable to remove the protecting groups and hydrolyse the -CO2R4 group to a CO2H group in a single step using one or more reagents in one-pot, for example wherein the deprotection reaction conditions require treating compound (11) with an aqueous solution of acid or base, such that the protecting groups are hydrolysed to reveal the corresponding alcohols and the CO2R4 group is hydrolysed to CO2H.
In one embodiment, the compound of formula (11) is treated with aqueous potassium hydroxide at reflux temperature to provide deoxycholic acid.
In a further embodiment, in the process described above, the compound of formula (6) is prepared by reacting a compound of formula (5) with an oxidising agent:
(5) (6) wherein PG is as defined hereinbefore for the compounds of formula (6) and (7).
The oxidising agent may be any agent suitable for the oxidation of secondary alcohols to ketones in the presence of an alkene group.
For example, the oxidising agent may be Dess-Martin periodinane, TEMPO (2, 2,6,6- tetramethylpiperidinyloxy) or the oxidising agent generated by combining (COCI)2 with DMSO or trifluoroacetic anhydride (TFAA) with DMSO i.e. the Swern oxidation, see Strategic Applications of Named Reactions in Organic Synthesis (L. Kurti and B. Czako, 1st Edition; Elsevier, 2005).
The use of the Swern oxidation is particularly advantageous because it does not require the utilisation of expensive or toxic reagents that are typically required for such oxidative transformations, such as chromium salts.
In one embodiment, the compound of formula (6) is prepared by reacting the compound of formula (5) with an oxidising agent, for example with a mixture of (COCI)2 or TFAA (trifluoroacetic anhydride), DMSO and triethylamine.
In one embodiment, the compound of formula (6) is prepared by reacting the compound of formula (5) with an oxidising agent, for example with a mixture of (COCI)2, DMSO and triethylamine, wherein the reaction is conducted at -70°C. In particular, the reaction solvent is dichloromethane.
In one embodiment, the compound of formula (6) is prepared by reacting the compound of formula (5) with an oxidising agent, for example with a mixture of TFAA, DMSO and triethylamine, wherein the reaction is conducted at between -20°C to -10°C, such as -15°C. In particular, the reaction solvent is dichloromethane.
The use of the Swern oxidation employing TFAA as the DMSO-activating agent was found to be particularly advantageous, because in addition to not requiring the utilisation of expensive or toxic reagents, it was found that the reaction proceeded cleanly and effectively at -20°C to -10°C, such as -15°C. In a further embodiment, in the process described herein, the compound of formula (5) is prepared by reacting a compound of formula (4) with an ethyltriphenylphosphonium salt: wherein PG is as defined hereinbefore for the compounds of formula (5), (6) and (7).
In one embodiment, the ethyltriphenylphosphonium salt is an ethyltriphenylphosphonium halide salt, such as ethyltriphenylphosphonium chloride, bromide or iodide.
In one embodiment, the compound of formula (5) is prepared by reacting the compound of formula (4) with an ethyltriphenylphosphonium salt, such as ethyltriphenylphosphonium bromide or iodide in the presence of a base, such as sodium bis(trimethylsilyl)amide (NaHMDS), lithium diisopropylamide (LDA), butyllithium (BuLi) or potassium tert-butoxide (KO‘Bu). In particular, the ethyltriphenylphosphonium salt is ethyltriphenylphosphonium bromide, and the base is potassium tert-butoxide.
The solvent may be THF, methyl-THF, dioxane, diethyl ether, dimethoxyethane, or methyl tert-butyl ether.
In particular, the solvent is THF.
In one embodiment, the solvent is THF and the reaction is conducted at a temperature of between 15 and 30°C.
This transformation is a Wittig reaction. This reaction advantageously installs the alkene with predominantly Z geometry in the compound of formula (5), which is the geometry required for fixing the stereochemistry of the methyl group in the compound of formula (10), during the step of reacting the compound of formula (9) with a compound of formula (b) in the presence of a coupling agent as described hereinbefore. In one embodiment, the solvent is THF and the reaction is conducted at a temperature of between 15 and 30°C. Advantageously, under these conditions, the compound of formula (5) is obtained with a E/Z ratio of 5:95. In a further embodiment, in the process described herein, the compound of formula (4) is prepared by reacting a compound of formula (3) with PG-X: wherein PG is as defined hereinbefore for the compounds of formula (4), (5), (6) and (7), and X is a leaving group.
As noted hereinbefore, methods of protecting functional groups, can be found in Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).
For example, for the compound of formula (4), the PG is selected from methoxymethyl (MOM) ether, benzyloxymethyl (BOM) ether, tetrahydropyranyl (THP) ether, tert-butyl ether fBu), allyl ether, paramethoxyphenyl (PMP) ether, benzyl ether (Bn), para-methoxybenzyl (PMB) ether, triphenylmethyl ether (Trit), -SiRs ether wherein R = C1-6 alkyl and/or aryl, trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert-butyldi methylsilyl (TBS) ether, tertbutyldiphenylsilyl (TBDPS) ether, Ci-e alkyl ester, acetate ester (Ac), pivaloate ester (Piv), benzoate ester (Bz), picolinate ester (Pico), methyl carbonate, ethyl carbonate, tert-butyl carbonate (Boc), allyl carbonate (Alloc), benzyl carbonate or N-phenyl carbamate.
For example, the compound of formula (4) may be prepared by reacting the compound of formula (3) with chloromethyl methyl ether (MOM), benzyl chloromethyl ether (BOM), 3,4-dihydropyran (THP), isobutylene (‘Bu), allyl bromide, 4-methoxyphenol or 4-iodoanisole (PMP), benzyl chloride (Bn), para methoxybenzyl chloride (PMB), trityl chloride (Trit), -SiRs halide (SiRs), trimethylsilyl chloride (TMS), triethylsilyl chloride (TES), triisopropylsilyl chloride (TIPS), chloro(dimethyl)isopropylsilane (DEIPS), tert-butyldimethylsilyl chloride (TBS), tert-butyldiphenylchlorosilane (TBDPS), Ci-s alkyl halide (C1-6 alkyl ester), acetyl chloride (Ac), pivaloyl chloride (Piv), benzoyl chloride (Bz), picolinoyl chloride (Pico), tosyl chloride (Ts), chloromethyl methyl carbonate, chloromethyl ethyl carbonate, di-tert-butyl carbonate (Boc), vinyl chloroformate, benzyl chloroformate) or N-phenyl carbamoyl chloride in the presence of a base.
In a particular embodiment, PG is a silyl ether protecting group, for example trimethylsilyl (TMS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS ether), diethylisopropylsilyl (DEIPS) ether, tert- butyldimethylsilyl (TBS) ether or tert-butyldiphenylsilyl (TBDPS) ether.
In particular, PG is tert-butyldimethylsilyl (TBS) ether, which can be installed by reacting the compound of formula (3) with tert-butyldimethylsilyl chloride and a base, for example imidazole. In one embodiment, the solvent is dichloromethane, and the reaction is conducted at temperature of between 15 and 30°C.
The selective incorporation of PG onto the C3 hydroxy group is advantageous because it allows the compound of formula (5) to be more easily purified following the reaction with the ethyltriphenylphosphonium salt, and in addition, it allows the compound of formula (6) to be prepared, because the C12 hydroxy group is the only alcohol group able to undergo oxidation.
In a further embodiment, in the process described herein, the compound of formula (3) is prepared by reacting a compound of formula (2) with an oxidising agent and a source of copper, and then hydrolysis of the imine to provide the compound of formula (3):
(2) (3) wherein R1 is selected from C1-6 alkyl or hydrogen;
R2 and R3 are selected from Ci-s alkyl or hydrogen; and n is selected from 0 or 1
The imine may be hydrolysed according to known procedures, for example using aqueous acid, such as 1 N hydrochloric acid.
In one embodiment, R1 is selected from methyl or hydrogen, and n is the selected from 0 or 1 .
In one embodiment, R2 and R3 are both hydrogen, or R2 is hydrogen and R3 is methyl, or R2 is methyl and R3 is hydrogen, or R2 is hydrogen and R3 is isopropyl, or R2 is isopropyl and R3 is hydrogen.
In a further embodiment, R1 is selected from methyl or hydrogen, R2 is hydrogen and R3 is methyl, or R2 is methyl and R3 is hydrogen, or R2 is hydrogen and R3 is isopropyl, or R2 is isopropyl and R3 is hydrogen and n is 0 or 1 , in particular n is 0.
In a particular embodiment, R1 is selected from methyl or hydrogen, R2 and R3 are both hydrogen and n is 0 or 1 , in particular n is 0. In particular, R1 is hydrogen. The oxidative transformation described hereinbefore is an example of a Schonecker-Baran Oxidation (see B. Schonecker et al., ACIE 2003, 42, 3240-3244; P.S. Baran et al., JACS 2015, 137, 13776-13779; T. Mu et al., Nature Communications 2020, 11 , 4371 and S. D. Offei et al., Steroids 2022, 186, 109088).
In one embodiment, the oxidising agent is a peroxide species such as hydrogen peroxide (H2O2), or is molecular oxygen (O2), and the source of copper is a divalent copper species. In particular, the source of copper is copper nitrate (Cu(NC>3)2) or a hydrate thereof, such as copper nitrate trihydrate (Cu(NO3)2.3H2O). Alternatively, the copper source may be Cu(MeCN)4PFs or Cu(Otf)2.
In a further embodiment, the oxidising agent is molecular oxygen (O2), and the source of copper selected from Cu(MeCN)4PFs and Cu(0tf)2. The reaction may also proceed in the presence of a reductant such as benzoin with triethylamine, FeBr2, Zn powder, EtsSiH or sodium ascorbate. In particular, the solvent is acetone or a 1 :1 mixture of acetone and methanol.
In a further embodiment, R1 is selected from methyl or hydrogen, R2 and R3 are both hydrogen, or R2 is hydrogen and R3 is methyl, or R2 is methyl and R3 is hydrogen, or R2 is hydrogen and R3 is isopropyl, or R2 is isopropyl and R3 is hydrogen and n is 0 or 1 , in particular n is 0, the oxidising agent is molecular oxygen, the source of copper is Cu(MeCN)4PFe or Cu(Otf)2, the reductant is sodium ascorbate, the solvent is a 1 :1 mixture of acetone and methanol, and the reaction is conducted at a temperature of between 20 and 70°C, in particular 50°C. In particular, R1 is hydrogen, R2 and R3 are both hydrogen and n is 0.
In one embodiment, the oxidising agent is a peroxide species, such as hydrogen peroxide (H2O2) and the source of copper is copper nitrate trihydrate (Cu(NO3)2.3H2O). In a particular embodiment, R1 is selected from methyl or hydrogen, R2 and R3 are both hydrogen, or R2 is hydrogen and R3 is methyl, or R2 is methyl and R3 is hydrogen, or R2 is hydrogen and R3 is isopropyl, or R2 is isopropyl and R3 is hydrogen and n is 0 or 1 , in particular n is 0, the oxidising agent is a peroxide species, such as hydrogen peroxide (H2O2) and the source of copper is copper nitrate trihydrate (Cu(NO3)2.3H2O), the solvent is THF and the reaction is conducted at temperature of between 15 and 30°C. In particular, R1 is hydrogen, R2 and R3 are both hydrogen and n is 0.
In a further embodiment, in the process described herein, the compound of formula (2) is prepared by reacting a compound of formula (1) with an amino pyridine derivative of formula (a):
wherein R1, R2, R3 and n are as defined hereinbefore for the compound of formula (2).
As noted hereinbefore, a particular amino pyridine derivative of formula (a) is wherein R1 is methyl or hydrogen, R2 and R3 are both hydrogen and n is 0, for example, an amino pyridine derivative of formula (a1) or (a2):
(a1) (a2)
The amino pyridine derivative of formula (a1) may be prepared by reducing 4-methyl-2-pyridinecarbonitrile with lithium aluminium hydride (LiAIF ) (see P.S. Baran et al., JACS 2015, 137, 13776-13779).
The amino pyridine derivative of formula (a2) is commercially available or may be obtained from commercially available starting materials and prepared from literature procedures.
The reaction of the compound of formula (1) with the amino pyridine derivative of formula (a), such as (a1) or (a2) may proceed under any conditions known in the art suitable for generating an imine species (i.e. a compound of formula (2)) from a compound containing a ketone.
In one embodiment, the compound of formula (1) and the amino pyridine derivative of formula (a), such as (a1) or (a2) are mixed under dehydrating conditions, for example with p-toluenesulfonic acid monohydrate and molecular sieves (i.e. 4 A molecular sieves) and heated under reflux conditions in a suitable solvent.
In one embodiment, the compound of formula (2) is prepared by reacting a compound of formula (1) with an amino pyridine derivative of formula (a), such as (a1) or (a2) in the presence of p-toluenesulfonic acid monohydrate or molecular sieves and a toluene solvent, and heating the reaction mixture to reflux in a Dean-Stark apparatus. In one embodiment, a compound of formula (2a) is prepared by reacting the compound of formula (1) with an amino pyridine derivative of formula (a2):
(1) (2a) in the presence of p-toluenesulfonic acid.
The present disclosure also provides the use of a compound of formula (1) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof:
The compound of formula (1) is known as etiocholanolone. It is commercially available from chemical suppliers. The use of etiocholanolone in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof is particularly beneficial because the stereochemistry required at the C3 and C5 of deoxycholic acid is already contained within etiocholanolone:
Consequently, this embodiment provides a comparatively simplified synthesis because the number of synthetic steps and/or comparative complexity of the chemistry required to access deoxycholic acid from other scaffolds (wherein the stereochemistry required at the C3 and C5of deoxycholic acid is not contained) are reduced.
In one embodiment, the present disclosure provides the use of a compound of formula (1) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, wherein the compound of formula (2) is prepared by reacting the compound of formula (1) with an amino pyridine derivative of formula (a): wherein R1, R2 and R3 are as defined hereinbefore.
In a further embodiment, the present disclosure provides the use of a compound of formula (1) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, wherein the compound of formula (2a) is prepared by reacting the compound of formula (1) with an amino pyridine derivative of formula (a2):
(1) (2a)
The present disclosure also provides a process for preparing a compound of formula (7), comprising the step of reacting a compound of formula (4) with an ethyltriphenylphosphonium salt to prepare the compound of formula (5):
and the step reacting the compound of formula (5) with an oxidising agent to prepare the compound of formula (6):
(5) (6) and the step of reacting a compound of formula (6) with a reducing agent to provide a compound of formula (7):
(6) (7) wherein PG is a protecting group as defined hereinbefore for the compound of formula (4), (5), (6) and (7).
This process advantageously combines the beneficial effects described hereinbefore associated with the Wittig reaction, the oxidation reaction, and the reduction reaction.
The present disclosure also provides a process for preparing a compound of formula (8), comprising the step of reacting a compound of formula (6) with a reducing agent to provide a compound of formula (7):
(6) (7) wherein PG is a protecting group, and the step of deprotecting the compound of formula (7) to provide the compound of formula (8):
(7) (8) wherein PG, the reducing agent and the deprotecting conditions are as defined hereinbefore for the compounds of formula (6) and (7).
The present disclosure also provides a process for preparing a compound of formula (8), comprising the step of deprotecting a compound of formula (7) to provide a compound of formula (8):
(7) (8) wherein PG is as defined hereinbefore for the compound of formula (7).
In a further embodiment, the present disclosure provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, comprising the step of preparing a compound of formula (7) by reacting a compound of formula (6) with a reducing agent (step (vi)) and one or more of steps (i)-(xii):
wherein R1, R2, R3, n, R4, and PG are as defined hereinbefore and Z+ is an organic cation, inorganic cation or metal cation.
Step (i) is a imine formation; step (ii) is an oxidation reaction; step (iii) is a selective reaction to protect a hydroxy group; step (iv) is a Wittig reaction; step (v) is an oxidation reaction; step (vi) is a reduction reaction; step (vii) is a deprotection reaction; step (viii) is a reaction to protect two hydroxy groups; step (ix) is an ene or Alder-ene reaction; step (x) is a reduction reaction; step (xi) is a deprotection/hydrolysis reaction; and step (xii) is a salt formation reaction as described hereinbelow.
In a further embodiment, the present disclosure provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, comprising the step of preparing a compound of formula (7a) by reacting a compound of formula (6a) with a reducing agent (step (vi)) and one or more of steps (i)-(xii): Step (i) is a imine formation; step (ii) is an oxidation reaction; step (iii) is a selective TBS protection reaction; step (iv) is a Wittig reaction; step (v) is a Swern oxidation reaction; step (vi) is a reduction reaction; step (vii) is a TBS deprotection reaction; step (viii) is an acetylation reaction (to protect two hydroxy groups); step (ix) is an ene or Alder-ene reaction; step (x) is a reduction reaction; step (xi) is a deprotection/hydrolysis reaction; and step (xii) is a sodium salt formation reaction as described hereinbelow.
In one embodiment, the present disclosure provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, comprising the step of reacting a compound of formula (6a) with a reducing agent to provide a compound of formula (7a), and deprotecting the compound of formula (7a) to provide a compound of formula (8). The reducing agent typically contains a source of hydride (H“), for example a metal hydride, or metal borohydride reagent, in particular the reducing agent is lithium tri-tert-butoxyaluminium hydride or lithium aluminium hydride. For the deprotection step, the deprotecting conditions are preferably suitable to remove the TBS group to provide the compound of formula (8). For example, the deprotection reaction requires treating the compound of formula (7a) with tetra-n-butylammonium fluoride (TBAF) in an appropriate solvent, such as tetrahydrofuran (THF).
In one embodiment, the present disclosure provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, comprising the step of deprotecting a compound of formula (7a) to provide a compound of formula (8).
Process for preparing pharmaceutically acceptable salts of deoxycholic acid In a further embodiment, the process comprises the step of converting deoxycholic acid into a pharmaceutically acceptable salt of deoxycholic acid (formula II): wherein Z+ is an organic cation, inorganic cation or metal cation.
The salts of the present disclosure can be synthesised from the parent compound (deoxycholic acid) by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid form of deoxycholic acid with the appropriate base in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
Specifically, the carboxylic acid moiety of deoxycholic acid may be anionic (e.g., -COOH may be -COO ), and as such, a salt may be formed with an organic or inorganic base, generating a suitable cation.
Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+ and K+, alkaline earth metal cations such as Ca2+ and Mg2+, and other cations such as Al3+ or Zn2+. Examples of suitable organic and inorganic cations include, but are not limited to, ammonium ion (i.e., NF ) and substituted ammonium ions (e.g., NHaR+, NH2R2+, NHRs+, NR4+). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
In a further embodiment, the process comprises the step of converting deoxycholic acid to the sodium salt of deoxycholic acid (formula III):
As described hereinbefore, the sodium salt can be synthesised from the parent compound (deoxycholic acid) by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.
In one embodiment, the step of converting deoxycholic acid to the sodium salt of deoxycholic acid comprises treating deoxycholic acid with sodium hydroxide, in particular 1 N aqueous sodium hydroxide.
In one embodiment, the step of converting deoxycholic acid to the sodium salt of deoxycholic acid comprises treating deoxycholic acid with 1 N sodium hydroxide, and heating the mixture to a temperature of between 70 and 100°C, in particular 90°C. Deoxycholic acid prepared by the process of the present disclosure
The present disclosure also provides a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, wherein the process comprises any of the process steps or processes described herein.
The present disclosure also provides deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes defined hereinbefore.
The present disclosure also provides deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by all the process steps defined hereinbefore.
The products obtainable by any of the process steps or processes defined hereinbefore are different to the products obtainable by the processes of the prior art, as demonstrated by their degree of purity.
The deoxycholic acid prepared by the process of the present disclosure is a synthetic deoxycholic acid free of any human or animal-derived substance as the process of the present disclosure relies on the use of non-animal derived starting materials and reagents.
Uses of deoxycholic acid and pharmaceutically acceptable salts thereof
As described hereinbefore, deoxycholic acid has numerous applications in both the pharmaceutical, vaccine and cosmetic industries.
In one embodiment, the present disclosure provides a pharmaceutical, immunogenic or cosmetic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to any of the processes described herein. In a particular embodiment, the present disclosure provides a pharmaceutical or immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to any of the processes described herein. In a further particular embodiment, the present disclosure provides an immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to any of the processes described herein. In any of such embodiments the pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance.
In a further embodiment, the present disclosure provides a pharmaceutical, immunogenic or cosmetic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein. In a particular embodiment, the present disclosure provides a pharmaceutical or immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein. In a further particular embodiment, the present disclosure provides an immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein. In any of such embodiments the pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance.
The pharmaceutical, immunogenic or cosmetic compositions typically comprise deoxycholic acid or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients or cosmetically acceptable carriers. In a particular embodiment, the immunogenic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to/obtainable by any of the processes described herein is a vaccine. The pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance. In the pharmaceutical, vaccine and cosmetic industries, deoxycholic acid or a pharmaceutically acceptable salt is sometimes used as: - Solubilizing agent: It can help dissolve certain compounds that are poorly soluble in water.
Detergent agent: It can be used to lyse or disrupt cell membranes (for instance bacterial cell wall or membrane) or viral particles, which can be useful in certain pharmaceutical or vaccine production processes. For instance, polysaccharide or conjugate vaccines such as against Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi, induce protective host immune responses against specific capsular polysaccharides. The manufacturing processes of such vaccines involve basic steps comprising of a) preparing a fermentation culture of bacterial cells that produce capsular polysaccharides b) lysing the bacterial cells in said fermentation culture and c) isolation of capsular polysaccharides from the lysed culture. The lysis of the cells is an important step at the end of the fermentation and is accomplished by the use of surfactants, such as deoxycholic acid or a pharmaceutically acceptable salt (such as sodium salt known as deoxycholate). Another example is influenza virus particles disruption for vaccine production (see for instance W.G. Laver et al., Postgrad Med J. 1976, 52(608), 373-8).
Reagent to precipitate a component of a composition, for instance an unconjugated free polysaccharide from carrier protein-bound material (see for instance Q.P. Lei et al., Developments in Biologicals 2000, 103, 259-264).
Adjuvant or adjuvant component: In some cases, it can be used as an adjuvant to enhance the immune response to a vaccine (see for instance G.J. Gorse, Vaccine 1995, 13(2), 209-214).
Component of a delivery system (see for instance J.S. Wilkhu et al., J Drug Target 2013, 21 (3):291 - 299.) - Cell culture medium component, for instance for bacteria culture (see for instance R. McKenzie et al., Vaccine 2006, 24(18), 3735-3745).
Antimicrobial (see for instance V.V.H. Le et al., BMC Microbiol. 2020, 20(1), 5).
Therefore, in one embodiment, the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable according to any one of the processes described herein in the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition. In a particular embodiment, the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable according to any one of the processes described herein in the manufacturing process of a pharmaceutical or immunogenic composition. In a further particular embodiment, the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable according to any one of the processes described herein in the manufacturing process of an immunogenic composition. The immunogenic composition may be a vaccine. In any of such uses, the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial. In any of such embodiments the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance.
In one such application, deoxycholic acid is used as a detergent for the extraction of complexes comprising lipid(s) and protein(s) from bacteria, for instance proteoliposomes, proteasomes, proteolipidic vesicles, etc. Complexes comprising lipid(s) and protein(s) are complexes that comprise at least one protein and at least one lipid and that may comprise more than one lipid and/or protein. In one such application, deoxycholic acid is used as a detergent for the extraction of outer membrane vesicles (OMVs).
Therefore, in one embodiment, the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to any of the processes described herein for extracting outer membrane vesicles from Gram-negative bacteria.
In a further embodiment, the present disclosure provides the use of deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein for extracting outer membrane vesicles from Gram-negative bacteria.
Although pharmaceutical grade bile acid preparations containing deoxycholic acid are commercially available at low cost from animal carcasses, there is a risk and concern that animal-derived bile acid products may contain animal pathogens and other harmful agents such as animal or microbial metabolites and toxins, including bacterial toxins such as pyrogens. Consequently, such animal-derived products must be subjected to expensive purification processes.
It is therefore beneficial to use the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the processes described herein (which relies on the use of non-animal derived starting materials and reagents) for extracting outer membrane vesicles. One embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for extracting complexes comprising lipid(s) and protein(s) from bacteria, such as proteoliposomes, proteasomes, or proteolipidic vesicles. Complexes comprising lipid(s) and protein(s) are complexes that comprise at least one protein and at least one lipid and that may comprise more than one lipid and/or protein. One embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for extracting outer membrane vesicles from Gram-negative bacteria, in particular from Neisseria meningitidis. One embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for use in extracting outer membrane vesicles from Gram-negative bacteria, in particular from Neisseria meningitidis serogroup B (MenB).
One further embodiment relates to the use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein for use in extracting outer membrane vesicles from Gram-negative bacteria, in particular from Neisseria meningitidis serogroup B (MenB). One further embodiment provides a composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein and an outer membrane vesicle (OMV). In particular, an outer membrane vesicle (OMV) from Neisseria meningitidis, in particular from Neisseria meningitidis serogroup B (MenB). Such compositions may be pharmaceutical or immunogenic compositions. The pharmaceutical or immunogenic composition typically further comprises one or more pharmaceutically acceptable excipients. The immunogenic composition may be a vaccine.
In any of such embodiments the extraction process may be free of any human or animal-derived substance. One embodiment relates to the use of a compound of formula (1), (2), (2a), (3), (4), (4a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10), (10a), (11) or (11 a) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof. A particular embodiment relates to the use of a compound of formula (1), (2), (2a), (3), (4), (4a), (5), (5a), (6), (6a), (7), (7a), (9), (9a), (10), (10a), (11) or (11 a) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof.
Another embodiment relates to a process for manufacturing a pharmaceutical, immunogenic or cosmetic composition comprising at least one step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein. In a particular embodiment, the present disclosure provides a manufacturing process of a pharmaceutical or immunogenic composition comprising at least a step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein. In a further particular embodiment, the present disclosure provides a manufacturing process of an immunogenic composition comprising at least a step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein. The immunogenic composition may be a vaccine. In any of such manufacturing processes the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial. In any of such embodiments the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance.
Another embodiment relates to a process for extracting complexes comprising lipid(s) and protein(s) from bacteria, such as proteoliposomes, proteasomes, or proteolipidic vesicles, using deoxycholic acid or a pharmaceutically acceptable salt prepared/obtainable by any of the processes described herein, or using the deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein. Complexes comprising lipid(s) and protein(s) are complexes that comprise at least one protein and at least one lipid and that may comprise more than one lipid and/or protein.
Another embodiment relates to a process for extracting outer membrane vesicles from Gram-negative bacteria using deoxycholic acid or a pharmaceutically acceptable salt prepared/obtainable by any of the processes described herein, or using the deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein.
A particular embodiment relates to a process for extracting outer membrane vesicles from Neisseria meningitidis, in particular from Neisseria meningitidis serogroup B (MenB), using deoxycholic acid or a pharmaceutically acceptable salt prepared/obtainable by any of the processes described herein, or using the deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by any of the processes described herein.
In any of such embodiments the extraction process may be free of any human or animal-derived substance.
In particular, the process for extracting outer membrane vesicles from Gram-negative bacteria comprises the steps of: a) Cultivating bacterial cells; b) Collecting and/or concentrating the cultivated cells; c) Disrupting the outer membranes of the cultivated cells with synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein, and forming outer membrane vesicles; and d) Recovering the outer membrane vesicles.
In particular, the process for extracting outer membrane vesicles from Gram-negative bacteria or from Neisseria meningitidis or from Neisseria meningitidis serogroup B, comprises the steps of: a) Cultivating the bacterial cells; b) Collecting and/or concentrating the cultivated cells; c) Disrupting the outer membranes of the cultivated cells with synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof prepared/obtainable by any of the processes described herein, and forming outer membrane vesicles; and d) Recovering the outer membrane vesicles. In such embodiment the steps a) to d) may be free of any human or animal-derived substance.
Although pharmaceutical grade bile acid preparations containing deoxycholic acid are commercially available at low cost from animal carcasses, there is a risk and concern that animal-derived bile acid products may contain animal pathogens and other harmful agents such as animal or microbial metabolites and toxins, including bacterial toxins such as pyrogens.
Therefore, in one embodiment, the present disclosure provides the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance, in the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition. In a particular embodiment, the present disclosure provides the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance, in the manufacturing process of a pharmaceutical or immunogenic composition. In a further particular embodiment, the present disclosure provides the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance, in the manufacturing process of an immunogenic composition. The immunogenic composition may be a vaccine. In any of such embodiments the manufacturing process of the pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance. In any of such uses, the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial and/or the deoxycholic acid or a pharmaceutically acceptable salt is prepared/obtainable by any of the processes described herein. The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be a component of the final pharmaceutical, immunogenic or cosmetic composition but such manufacturing processes of a pharmaceutical, immunogenic or cosmetic composition using the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof are not a production process of the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof. The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. Therefore, in one embodiment, the present disclosure provides the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance, as a detergent for the extraction of complexes comprising lipid(s) and protein(s) from bacteria, for instance proteoliposomes, proteasomes, proteolipidic vesicles, etc. Complexes comprising lipid(s) and protein(s) are complexes that comprise at least one protein and at least one lipid and that may comprise more than one lipid and/or protein. In one such application, synthetic deoxycholic acid, in particular synthetic deoxycholic acid free of any human or animal-derived substance, is used as a detergent for the extraction of outer membrane vesicles (OMVs). The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. In any of such embodiments the extraction process may be free of any human or animal-derived substance.
In one embodiment, the present disclosure provides a composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof and complexes comprising at least one lipid and at least one protein extracted from bacteria. Such complexes may be proteoliposomes, proteasomes or proteolipidic vesicles.
One embodiment relates to the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance, for extracting outer membrane vesicles from Gramnegative bacteria, in particular from Neisseria meningitidis. One embodiment relates to the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt, in particular the use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance, for use in extracting outer membrane vesicles from Neisseria meningitidis serogroup B (MenB). The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. The extraction process may be free of any human or animal- derived substance.
In one embodiment, the present disclosure provides a composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance, and an outer membrane vesicle (OMV). In particular, the present disclosure provides a composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance, and an outer membrane vesicle (OMV) from Neisseria meningitidis, in particular from Neisseria meningitidis serogroup B (MenB). Such compositions may be pharmaceutical or immunogenic compositions. The pharmaceutical or immunogenic composition typically further comprises one or more pharmaceutically acceptable excipients. The immunogenic composition may be a vaccine. The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. The composition may be free of any human or animal-derived substance. Another embodiment relates to a process for manufacturing a pharmaceutical, immunogenic or cosmetic composition comprising at least one step using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance. In a particular embodiment, the present disclosure provides a process for manufacturing a pharmaceutical or immunogenic composition comprising at least one step using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal- derived substance. In a further particular embodiment, the present disclosure provides a process for manufacturing an immunogenic composition comprising at least one step using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, in particular a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance. The immunogenic composition may be a vaccine. In any of such manufacturing processes the deoxycholic acid or a pharmaceutically acceptable salt may be used as solubilizing agent, detergent or surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial and/or the deoxycholic acid or a pharmaceutically acceptable salt is prepared/obtainable by any of the processes described herein. The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be a component of the final pharmaceutical, immunogenic or cosmetic composition but such manufacturing processes of a pharmaceutical, immunogenic or cosmetic composition comprising a step using the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof are not a production process of the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof. In any of such embodiments the manufacturing process of the pharmaceutical, immunogenic or cosmetic composition may be free of any human or animal-derived substance.
Another embodiment relates to a process for extracting complexes comprising lipid(s) and protein(s) from bacteria, such as proteoliposomes, proteasomes, or proteolipidic vesicles, using a synthetic deoxycholic acid or a pharmaceutically acceptable salt, in particular using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance. Complexes comprising lipid(s) and protein(s) are complexes that comprise at least one protein and at least one lipid and that may comprise more than one lipid and/or protein. In particular, the process is for extracting outer membrane vesicles from Gram-negative bacteria. The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. The extraction process may be free of any human or animal-derived substance.
A particular embodiment relates to a process for extracting outer membrane vesicles from Gram-negative bacteria using a synthetic deoxycholic acid or a pharmaceutically acceptable salt, in particular using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal- derived substance. A particular embodiment relates to a process for extracting outer membrane vesicles from Neisseria meningitidis, in particular from Neisseria meningitidis serogroup B (MenB), using a synthetic deoxycholic acid or a pharmaceutically acceptable salt, in particular using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof, free of any human or animal-derived substance. The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein. The extraction process may be free of any human or animal-derived substance. In particular, the process for extracting outer membrane vesicles from Gram-negative bacteria comprises the steps of: a) Cultivating the bacterial cells b) Collecting and/or concentrating the cultivated cells c) Disrupting the outer membranes of the cultivated cells with synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof and forming outer membrane vesicles d) Recovering the outer membrane vesicles.
In particular, the process for extracting outer membrane vesicles from Gram-negative bacteria comprises the steps of: a) Cultivating bacterial cells b) Collecting and/or concentrating the cultivated cells c) Disrupting the outer membranes of the cultivated cells with synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance and forming outer membrane vesicles d) Recovering the outer membrane vesicles.
The synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof may be prepared according to any of the processes described herein.
In particular, the bacterial culture involves the use of materials free of any human or animal-derived substance. The steps a) to d) may be free of any human or animal-derived substance. The collection and/or concentration of the cultivated bacterial cells may be according to any methods known in the art.
In particular, the disrupting of the outer membranes of the cultivated bacterial cells with synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof free of any human or animal-derived substance is under heating, for example from about 50°C to about 60°C, or at about 56°C, and for a time ranging from about 10 to about 20 minutes, or at about 15 minutes.
A treatment with a nuclease, like benzonase, according to any methods known in the art can be used to digest nucleic acids after formation of the outer membrane vesicles and before recovery of the outer membrane vesicles.
The recovery of the outer membrane vesicles may be according to any methods known in the art. Recovered outer membrane vesicles may then be concentrated according to any methods known in the art, and optionally further purified according to any methods known in the art. A suitable method to obtain bacterial outer membrane vesicles may be as disclosed in Helting et al. (Acta Pathol Microbiol Scand C. 1981 Apr;89(2):69-78), in WO01/91788, in W02005004908 or in Example 2 of US 4,695,624.
Intermediate compounds
In one embodiment, the present disclosure also provides a compound of formula (2), (5), (6), (7), (8), (9), (10) or (11): wherein PG is as defined hereinbefore.
In a particular embodiment, the present disclosure also provides a compound of formula (6) or (7) wherein PG is as defined hereinbefore.
In a further embodiment, the present disclosure provides a compound of formula (2), (2a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10), (10a), (11) or (1 1a):
In a further embodiment, the present disclosure provides a compound of formula (2), (2a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10) or (10a).
In a further embodiment, the present disclosure provides a compound of formula (2), (2a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10), (10a), (11) or (1 1a): for use in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof.
EXAMPLES
The present disclosure will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples. In the examples, the following abbreviations are used.
Aq. aqueous
CDCb deuterated chloroform
DCM dichloromethane
DIPO diisopropyl ether
DMAP 4-(dimethylamino)pyridine
DMSO dimethyl sulfoxide
EDTA ethylenediaminetetraacetic acid
EtAICh ethylaluminium dichloride
EtaN triethylamine
EtOAc ethyl acetate
Et2<3 diethyl ether
EtPPhsBr ethyltriphenylphosphonium bromide
HCI hydrochloric acid
HPLC high pressure liquid chromatography
KOH potassium hydroxide
KO'Bu potassium terf-butoxide
LiHB(secBu)3 lithium tri-sec-butylborohydride (L-Selectride)
LTBA lithium tri-tert-butoxyaluminium hydride
MeCN acetonitrile
MeOD deuterated methanol
MeOH methanol
MgSO4 magnesium sulphate
NaaSaOs sodium metabisulphite
NMR nuclear magnetic resonance spectroscopy
PMA phosphomolybdic acid
Pd/C palladium on carbon
TBAF tetrabutylammonium fluoride
THF tetrahydrofuran
TLC thin layer chromatography
TBDMSCI tertbutyldimethylsilyl chloride
V volumes Synthetic methods
All starting materials and solvents were obtained either from commercial sources or prepared according to the literature citation. Unless otherwise stated all reactions were stirred. Organic solutions were routinely dried over anhydrous magnesium sulfate. Hydrogenations were performed on a Parr hydrogenator, a Thales H-cube flow reactor under the conditions stated or under a balloon of hydrogen. Normal phase column chromatography was routinely carried out on an automated flash chromatography system such as CombiFlash Companion or CombiFlash RF system using pre-packed silica (230-400 mesh, 40-63 pm) cartridges. NMR data
1H NMR spectra were acquired on a Bruker spectrometer at 300 MHz. Either the central peaks of chloroform-cf, methanol-c/4 or an internal standard of tetramethylsilane were used as references. For NMR data, where the number of protons assigned is less than the theoretical number of protons in the molecule, it is assumed that the apparently missing signal(s) is/are obscured by solvent and/or water peaks. In addition, where spectra were obtained in protic NMR solvents, exchange of NH and/or OH protons with solvent occurs and hence such signals are normally not observed.
HPLC analysis and purification
In the following examples a number of a compounds were analysed/purified by high performance liquid chromatography. In each case the samples were dissolved in acetonitrile prior to injection. The HPLC system included Waters Alliance 2996 PDA and/or Waters 2998 PDA (photodiode array detectors) and
Corona CAD (charged aerosol detector) from Thermofisher.
Compound 9a
Column: Waters X-Bridge C18, 4.6x150 mm, 5 pm
UV Detection: PDA 2998 210 nm
Column flow rate: 1 .0 mL/min
Column temperature: 40°C
Mobile phase: A: NH4COOH 5mM pH 8
B: acetonitrile
Injection volume: 5.0 pL Gradient method:
Compound I - deoxycholic acid
Column: X-Bridge C18, 4.6x150 mm, 5 m
Detection: Corona CAD
Column flow rate: 1 .0 mL/min
Column temperature: 40°C
Mobile phase: A: water +0.02% formic acid
B: acetonitrile
Injection volume: 5.0 pL Gradient method:
Compound III - deoxycholic acid sodium salt
Column: X-Bridge C18, 4.6x150 mm, 5 pm
Detection: Corona CAD
Column flow rate: 1 .0 mL/min
Column temperature 40°C
Mobile phase: A: water +0.02% formic acid
B: acetonitrile
Injection volume: 5.0 pL Gradient method:
Synthetic preparations
Compound 2a
To a vessel fitted with Dean Stark apparatus were added etiocholanolone (44 g, 151.5 mmol), toluene (528 mL, 12V), 2-picolylamine (78.3 mL, 757.4 mmol, 5 eq) and para-toluenesulfonic acid monohydrate (5.76 g, 30.3 mmol, 0.2 eq). The resulting solution was then refluxed for 5 hours. Reaction progress was monitored by NMR, and the reaction was considered complete when the signal between 2.17-1 .99 ppm was no longer detected. Upon completion of the reaction the heating was stopped, and the mixture was allowed to cool to room temperature. The mixture was then concentrated to dryness. The resulting residue was diluted with EtOAc (200 mL, 4.5V), and the organic phase was washed with water (3 x 100 mL, 3 x 2.3V). The organic phase was then separated, dried over MgSO4 and filtered. The filtrate was then concentrated to obtain a yellow residue (62.9 g). The residue was then triturated at 80°C for 1 hour in a 90:10 heptane/EtOAc mixture (3 to 3.2V). The resulting crystals were then separated to provide a beige crystalline solid (48.7 g, 85%), 1H NMR (300 MHz, CDCb): 5 8.51 (d, J = 5.0 Hz, 1 H), 7.65 (t, J = 7.9 Hz, 1 H), 7.41 (d, J = 7.9 Hz, 1 H), 7.13 (dd, J = 7.9, 5.0 Hz, 1 H), 4.70-4.50 (m, 2H), 3.71-3.65 (m, 1 H), 2.53- 2.36 (m, 1 H), 2.35-2.17 (m, 1 H), 0.96 (s, 3H), 0.88 (s, 3H) (traces of 2-picolylamine are detected in NMR).
Compound 3
To a reaction vessel were added compound 2a (48.7 g, 128.0 mmol) and THF (630 mL, 13V), followed by the addition of Cu(NO3)2.3H2O (34 g, 140.7 mmol, 1.1 eq) in one portion. The resulting heterogeneous mixture was stirred at room temperature for 30 minutes, and then at 50°C for 45 minutes. During the heating step, it was observed that the solid dissolved to first provide a dark blue solution and then a light blue precipitate. The suspension was then cooled to room temperature. Hydrogen peroxide 130 volumes
(35% by weight) (59.4 mL, 639.0 mmol, 5.0 equiv) was slowly added (exotherm and gas evolution was observed) at a speed such that the temperature of the reaction mixture did not exceed 31 °C (addition of hydrogen peroxide required approximately 20 minutes and also cooling of the reaction vessel with an ice bath). Upon complete addition of the hydrogen peroxide, the mixture was allowed to stir at room temperature for 19 hours. Following this, an aqueous solution of Na4EDTA (107.2 g in 490 mL of water, 2.2 eq) and EtOAc (500 mL, 10V) were added to the reaction mixture, and the mixture was allowed to stir for at least 20 hours at room temperature. After which, the phases were separated and the organic phase was recovered. The organic phase (1) was then washed with 1 N HCI (2 x 250 mL, 2 x 5V). The acidic aqueous phase (product 3 was detected in this phase) was extracted with EtOAc (3 x 100 mL, 3 x 2V) to provide organic phase (2). The combined organic phases (1) + (2) were then washed with a concentrated aqueous solution of sodium metabisulphite (Na2S20s) (250 g in 500 mL of water), and the presence of peroxide in the organic phase was checked using peroxide test strips. The combined organic phases were continually washed with the aqueous solution of sodium metabisulphite until the peroxide test strip indicated a concentration < 20 ppm. The combined organic phase was then dried over MgSO4 and filtered. The filtrate was then concentrated to obtain a yellow solid (34.9 g). The solid was then triturated at room temperature for 1 hour in isopropyl ether (175 ml, 5V). The solid was then filtered and rinsed with isopropyl ether (2 X 1 V) to provide a beige solid (28.7 g, 73%), 1H NMR (300 MHz, CDCh): 5 3.82-3.71 (m, 1 H), 3.70- 3.56 (m, 1 H), 2.52-2.37 (m, 1 H), 2.19-2.05 (m, 1 H), 0.94 (s, 3H), 0.91 (s, 3H).
Compound 4a
To a reaction vessel under an atmosphere of nitrogen were added compound 3 (31 g, 101 .3 mmol), DCM (465 mL, 15V) and imidazole (14.5 g, 212.7 mmol, 2.1 eq). Tertbutyldimethylsilyl chloride (TBDMSCI) (18.3 g, 121 .6 mmol, 1 .2 eq) was added thereto, and the resulting mixture was allowed to stir at room temperature for 4 hours. A precipitate was observed shortly after the addition of TBDMSCI. The reaction was monitored by TLC (heptane/EtOAc 50:50, PMA stain). Upon completion of the reaction, the reaction mixture was washed with water. The organic phase was then dried over MgSO4 and concentrated to dryness. The resulting residue was triturated in a minimum volume of heptane and filtered to provide compound 4a (31 .9 g). The 12.5 g of crude product obtained by evaporation of the mother liquors was purified by automatic column chromatography (silica column: 120 g eluent: 100% heptane to heptane/DIPO 8:2 in 20 CV). The product-containing fractions were combined and concentrated to dryness to provide a further compound 4a (6.9 g). In total, from trituration and column chromatography, 38.8 g (31 .9 g + 6.9 g) of compound 4a were obtained with a yield of 91 %, 1H NMR (300 MHz, CDCb): 6 3.83-3.70 (m, 1 H), 3.67-3.49 (m, 1 H),2.98 (brs, 1 H), 2.53-2.38 (m, 1 H), 2.20-2.02 (m , 1 H), 0.93 (s, 3H), 0.91 (s, 3H), 0.88 (s, 9H), 0.05 (s, 6H).
Compound 5a
To a reaction vessel was added EtPPhaBr (1 19.8 g, 322.7 mmol, 3.5 eq) and the vessel was dried under vacuum at 70°C for 1 hour. The vessel was then placed under a nitrogen atmosphere, and THF (500 ml_, 12.9V) was added thereto at room temperature. A solution of t-BuOK (36.4 g, 322.7 mmol, 3.5 eq) in THF (500 ml_, 12.9V) was then very quickly added dropwise thereto (reaction mixture turns orange) at room temperature, and the mixture was allowed to stir for 1 hour. A solution of compound 4a (38.8 g, 92.2 mmol, 3.5 eq) in THF (500 mL, 12.9V) was added thereto and the resulting mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC (heptane/DIPO 80:20, PMA stain). Upon completion of the reaction, the reaction mixture was poured onto ice (1.5L). The aqueous phase was extracted with isopropyl ether (2 x 1 L, 2 x 25.8V) and the organic phase separated. The organic phase was then dried over MgSCh, filtered and concentrated, The resulting residue was then triturated with heptane (4V), filtered and washed with heptane/isopropyl ether 9/1 to recover the product (whilst removing as much triphenylphosphine oxide as possible). Following the trituration and wash processes, the resultant crude product was further purified by column chromatography (eluent: heptane/isopropyl ether 9/1 , approximately 40 CV) to provide pure product (25.9 g, 65%, E/Z 5:95), 1H NMR (300 MHz, CDCb): 5 5.28-5.13 (m, 1 H), 3.85-3.70 (m, 1 H), 3.67-3.50 (m, 1 H), 2.54-2.33 (m, 1 H), 2.27-2.07 (m, 1 H), 0.91 (s, 3H), 0.88 (s, 9H), 0.84 (s, 3H), 0.05 (s, 6H).
Compound 6a - (COCI)? DMSO activation
To an oven-dried reaction vessel under an atmosphere of nitrogen, DCM (145 ml_, 11V) was added and cooled to -78° C with an ice/acetone bath. Oxalyl chloride (2.83 ml_, 33 mmol, 1.1 eq) was first introduced into the reaction vessel, followed by the dropwise addition of DMSO (5.1 ml_, 72 mmol, 2.2 eq) diluted in DCM (2 ml_). Following the introduction of DMSO, an exotherm of +10°C was observed. The reaction mixture was allowed to stir at < -60°C for at least 10 minutes, after which point a solution of compound 5a (13 g, 30 mmol) in DCM (30 ml_, 2.3V) was added dropwise thereto at the same temperature. During and following the addition of compound 5a a further exotherm was observed. The reaction mixture was allowed to stir at a temperature <-60° C for 2 hours, after which point triethylamine (22.5 mL, 162 mmol, 5.4 eq) was added dropwise thereto. During and following the addition of triethylamine a further exotherm was observed. The reaction mixture was stirred and allowed to warm to room temperature over a duration of 16.5 hours. The reaction was monitored by TLC (heptane/DIPO 90:10, PMA stain). Upon completion of the reaction, water 100 mL (7.7V) was added and the organic phase was separated. The organic phase was then washed with water (2 x 50 mL, 2 x 3.8V), dried over MgSO4 and filtered. The organic phase was then concentrated to dryness to provide an off-white solid (12.8 g). The solid (12.8 g combined with two other batches of comparable purity, total mass: 24.5 g) was then triturated with heptane (2V) and separated to provide a white solid (21 g, 84%), 1H NMR (300 MHz, CDCh): 5 5.50-5.35 (m, 1 H), 3.65-3.45 (m, 1 H), 2.60-2.41 (m, 1 H), 2.39-2.21 (m, 2H), 2.18-2.03 (m, 1 H), 1.17 (s, 3H), 0.97 (s, 3H), 0.84 (s, 9H, 0.00 (s, 6H).
Compound 6a - trifluoroacetic anhydride (TFAA) DMSO activation
To a reaction vessel under an atmosphere of nitrogen were successively added, compound 5a (20 g, 46.2 mmol), THF (80 ml_) and DMSO (22 g, 282.2 mmol, 6.1 eq). The resulting solution was cooled to -15°C and trifluoroacetic anhydride (20 g, 95.2 mmol, 2.06 eq) in 10 mL of THF was added dropwise thereto over 60 minutes. The reaction mixture was allowed to stir an additional 15 minutes at -15°C, and then triethylamine (21 g, 207.5 mmol, 4.5 eq) in 10 mL of THF was added dropwise thereto over 2 hours at the same temperature. The reaction mixture was stirred for an additional hour at -15°C and then allowed to warm to room temperature over a duration of 12 hours. Upon completion, the reaction mixture was poured into 100 mL of water and extracted with diisopropyl ether (3 x 50 mL), the combined organic extracts were then washed with a saturated aqueous solution of ammonium chloride (3 x 40 mL), dried over MgSO4 and filtered. The organic phase was then concentrated in vacuo. The obtained semi-solid residue was then triturated with acetonitrile (40 mL), and filtered to provide a white solid (17.6 g, 88%) m.p.149°C; [a]D= +202.9° (CHCb, c=0.3g/100ml) 20°, purity HPLC = 97.8%; 1H NMR (300 MHz, CDCb): 5 5.50-5.35 (m, 1 H), 3.65-3.45 (m, 1 H), 2.60-2.41 (m, 1 H), 2.39-2.21 (m, 2H), 2.18-2.03 (m, 1 H), 1.17 (s, 3H), 0.97 (s, 3H), 0.84 (s, 9H, 0.00 (s, 6H).
Compound 7a - LiAIHfO’Buh reduction
To an oven-dried reaction vessel under an atmosphere of nitrogen were added compound 6a (19 g, 44.1 mmol) and THF (400 mL, 21V). The reaction mixture was then cooled to 0°C, and a solution of LiAIH(O*Bu)3 (56.3 g, 220.5 mmol, 5 eq) in THF (170 mL - including 50 mL for rinsing) was added. Upon addition of the hydride an exotherm was observed. The reaction mixture was allowed to stir at 0°C for 4 hours, and reaction progress was monitored by TLC (heptane/DIPO 90:10, PMA stain). Upon completion of the reaction the mixture was poured slowly over ice (1 L). EtOAc (50 mL, 2.6V) was also added, and the mixture was stirred for 17 hours. The reaction mixture was then filtered and rinsed with EtOAc. The filtrate was then decanted, and the organic phase was separated. The aqueous phase was then extracted with EtOAc (2 x 200 mL, 2 x 10.5V). The combined organic phases were dried over MgSO4 and filtered. The filtrate was then concentrated to dryness to provide a white solid (18.5 g, 97%). No further purification of the product was performed, and the product was directly engaged in the following step (deprotection of the TBS group), 1H NMR (300 MHz, CDCb): 6 5.42-5.24 (m, 1 H), 5.24-514 (m, 0.08H, Dia 12p), 4.44-430 (m, 1 H), 3.83-3.70 (m, 0.08H, Dia 12p), 3.68-3.50 (m, 1 H), 2.52-2.07 (m, 2H), 0.90 (s, 3H), 0.89 (s, 3H), 0.88 (s, 9H), 0.05 (s, 6H). Ratio 12a/12p = 92/8.
Compound 7a - UAIH4 reduction
To a reaction vessel under an atmosphere of nitrogen was added THF (150 mL), and lithium aluminium hydride (2 g, 52.6 mmol, 0.6 eq) in small portions at room temperature, at a speed such that the reaction temperature did not exceed 40°C. The reaction mixture was then cooled to 10°C, after which compound 6a (38.3 g, 89 mmol) (dissolved in THF (50 mL)) added dropwise thereto over 60 minutes at 10°C. The resulting mixture was then allowed to stir for 30 minutes at room temperature. The reaction was monitored by TLC (heptane/DIPO 90:10, PMA stain). Upon completion, the reaction mixture was cooled to 10°C, and then ethyl acetate (20 mL) was added dropwise over 30 minutes. The reaction mixture obtained was then poured onto crushed ice (200 g) and ethyl acetate (100 mL). The resulting mixture was then acidified to pH 3 with aqueous HCI 3N. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (3 x 50 mL). The combined organic phases were dried over MgSCM and filtered, and the filtrate was concentrated in vacuo. The resulting residue (solid-liquid mixture) was triturated with acetonitrile (2 x 100 mL) to provide a white solid (32.3 g, 84%); m.p. = 169°C; [a]D= +61 °(CHCb, c=0.3g/100ml) 20°C; Ratio 12a/12p = 96.7/3.3; 1H NMR (300 MHz, CDCb): 6 5.42-5.24 (m, 1 H), 5.24-514 (m, 0.08H, Dia 12p), 4.44- 430 (m, 1 H), 3.83-3.70 (m, 0.08H, Dia 12p), 3.68-3.50 (m, 1 H), 2.52-2.07 (m, 2H), 0.90 (s, 3H), 0.89 (s, 3H), 0.88 (s, 9H), 0.05 (s, 6H). Ratio 12a/12p = 92/8. Compound 8
To a reaction vessel under an atmosphere of nitrogen were added compound 7a (18.5 g, 42.8 mmol, 92% purity) and THF (39 mL, 2V). A solution of TBAF (1 N in THF, 171.2 mL, 4 eq) was then added at 25°C, and following addition the solution was heated to 65°C (reaction mixture was red). Reaction progress was monitored by TLC (heptane/DIPO 80:20, PMA stain). Upon completion of the reaction, the reaction mixture was poured into water (500 mL, 27V). The mixture was then decanted, and the organic phase was separated (yellow oil decanted). The aqueous phase was then extracted with EtOAc (3 x 100 mL, 3 x 5.4V). The combined organic phases were then dried over MgSC and filtered. The filtrate was then concentrated to dryness. The resulting residue (solid deposits in EtOAc) was then purified by column chromatography (eluent: heptane/EtOAc 8/2 (30CV), 7/3 (10CV) and EtOAc) to provide pure product (9.5 g, 70% - yield over two steps), 1H NMR (300 MHz, CDCh): 6 5.40-5.22 (m, 1 H), 4.44-4.30 (m, 1 H), 3.73-3.51 (m, 1 H), 2.48- 2.32-2.10 (m, 21 H), 0.92 (s, 3H), 0.89 (s, 3H), optical rotation: [a]D 25 C = + 65.9° (CHCI3, c = 0.3), mp: 181.5°C.
Compound 9a
To a reaction vessel under an atmosphere of nitrogen were added compound 8 (7.7 g, 24.2 mmol) and DCM (154 mL, 20V). Then, acetic anhydride (9 mL, 96.8 mmol, 4 eq), triethylamine (20 mL, 145.2 mmol, 6 eq) and DMAP (591 mg, 4.84 mmol, 0.2 eq) were successively added to the reaction, and the reaction mixture was allowed to stir for 16.5 hours at room temperature. Reaction progress was monitored by TLC (heptane/EtOAc 60:40, PMA stain). After 16.5 hours TLC analysis indicated presence of the monoacetate. Following this observation further acetic anhydride (2.3 mL, 24.2 mmol, 1 eq) and triethylamine (3.4 mL, 24.2 mmol, 1 eq) were added to the reaction mixture and the reaction was allowed to stir for an additional 2 hours. Upon completion of the reaction, the reaction mixture was directly evaporated to dryness. The remaining residue was dissolved in 50 ml_ of DIPO, and the solution was washed with water (3 x 50 ml_, 3 x 6.5V). The organic phase was dried over MgSCU and filtered. The filtrate was then concentrated to dryness to provide an off-white solid (9.91 g). The solid was then triturated with pentane (2 x 3 ml_, 2 x 0.4V) and drained to provide a white solid (8.3 g, 86%), 1H NMR (300 MHz, CDCb): 6 5.57-5.47 (m, 1 H), 5.29-5.13 (m, 1 H), 4.84-4.66 (m, 1 H), 2.48-2.17 (m, 2H), 2.10 (s, 3H), 2.06 (s, 3H), 1.00-0.92 (m, 6H), optical rotation - [O]D25 C = + 135.9° (CHCb, c = 0.6), mp: 132.4°C, HPLC purity: 95.5%. The product had a retention time of 20.9 minutes, detailed HPLC information is provided hereinabove. Compound 10a
To an oven-dried reaction vessel under an atmosphere of nitrogen were added DCM dried on molecular sieves (catalyst support, sodium Y zeolite) (190 mL, 44V), methyl propiolate (5.7 mL, 64.1 mmol, 6 eq) and a solution of EtAICb (1M in hexane, 64.1 mL, 64.1 mmol, 6 eq). The solution was stirred at room temperature for 30 minutes (clear yellow solution). After which, a solution of compound 9a (4.3 g, 10.7 mmol) in DCM dried on molecular sieves (20 mL, 4.7V) was added in one portion at room temperature. The reaction mixture was then allowed to stir for 2 days at room temperature. Reaction progress was monitored by NMR, and the reaction was considered complete when the NMR no longer contained signals between 5.50 and 5.44 ppm. Upon completion of the reaction, the reaction mixture was poured into water (750 mL, 174V) and a cloudy emulsion was observed. The mixture was allowed to stir until the cloudiness resolved. The mixture was then decanted, and the organic phase (1) was separated. The remaining aqueous phase was extracted with DCM (23V) and the organic phase was separated to provide organic phase 2. The combined organic phases (1 +2) were washed with a 1 N HCI solution (75 mL, 17.4V), dried over MgSO4 and filtered. The filtrate was then concentrated to dryness to provide a yellow oil (6.57 g). The yellow oil was then purified by manual column chromatography (silica: >20 parts, eluent: heptane/EtOAc 95:5 (10L), then 90:10 (2L)). The product-containing fractions were combined and concentrated to dryness to provide a white friable solid (3.8 g, 72% yield), 1H NMR (300 MHz, CDCb): 6 6.89-6.73 (m, 1 H), 5.90- 5.75 (m, 1 H), 5.59-5.46 (m, 1 H), 5.18-5.05 (m, 1 H), 4.82-4.60 (m, 1 H), 3.71 (s, 3H), 2.93-2.65 (m, 1 H), 2.48-2.17 (m, 2H), 2.08 (s, 3H), 2.03 (s, 3H), 0.92 (s, 3H), 0.79 (s, 3H). Compound 11 a
To an autoclave were added compound 10a (3.8 g, 78.1 mmol) and MeOH (1 14 mL, 30V). The autoclave was then purged with nitrogen (x3), and then Pd/C (10% with 50% H2O, 1.1 g, 28%wt) were added. The autoclave was then again purged with nitrogen (x3) and then purged with hydrogen (x3). The autoclave was then pressurized to 4 bars (hydrogen) and the reaction mixture was allowed to stir at room temperature for 16 hours. Reaction progress was monitored by NMR, and the reaction was considered complete when the NMR no longer contained the signals of the ethylenic protons (between 7.0 and 5.5 ppm). Upon completion of the reaction, the reaction mixture was filtered through celite, and the celite was rinsed with MeOH (1V) and the MeOH was concentrated to dryness. To remove any remaining insoluble particulate matter, the product was filtered through a silica plug and eluted with a heptane/EtOAc 8:2 mixture to provide a white solid after evaporation (3.6 g). The white solid was then recrystallised from heptane (3V) to provide a white solid (2.93 g, 77%), 1H NMR (300 MHz, CDCh): 6 5.14-5.05 (m, 1 H), 4.81-4.63 (m, 1 H), 3.70 (s, 3H), 2.93-2.65 (m, 1 H), 2.44-2.28 (m, 1 H), 2.10 (s, 3H), 2.03 (s, 3H), 1 .56 (s, 3H), 0.90 (s, 3H), 0.72 (s, 3H).
Compound I - deoxycholic acid
To a reaction vessel were added compound 11 a (3.3 g, 6.7 mmol), water (33 mL, 10V), methanol (100 mL, 30V) and KOH (6.7 g, 1 19.7 mmol, 17.8 eq). The reaction mixture was then heated at reflux for 20 hours. Reaction progress was monitored by NMR, and the reaction was considered complete when the NMR no longer contained signals at 5.14-5.05 and 4.81-4.63 ppm. Upon completion of the reaction, the methanol was evaporated, and water was added to the resulting residue. The aqueous solution was then washed with EtOAc (100 ml_, 30V). The organic phase was then acidified to pH=1 with a 3N HCI solution (which induced the formation of a precipitate). The precipitate was drained and rinsed with water. The precipitate was then taken up with 1 eq of 1 N NaOH and heated under reflux with stirring for 20 minutes. The solution was then cooled to 20°C and filtered through celite. The filtrate was then acidified to pH 1 , and the resulting suspension was stirred for 30 minutes. The precipitate was then drained, rinsed with water and dried under vacuum in the presence of P2O5. The solid obtained was then recrystallised from methyl ethyl ketone (20V) to provide (after drying under vacuum at 45°C) white crystals (1.8 g, 76%), 1H NMR (300 MHz, MeOD): 6 4.01-3.93 (m, 1 H), 3.62-3.47 (m, 1 H), 2.44-2.15 (m, 2H), 0.95 (s, 3H), 0.73 (s, 3H), optical rotation: [a]D 25 C = + 53.6° (MeOH, c = 0.2, literature: + 55° - Norman Arkiv foer Kemi, 1956, 8, 331), mp: 174.6°C (deoxycholic acid from Sigma Aldrich 99% mp: 170-175°C), HPLC purity 97.94%. The product had a retention time of 11 .1 minutes, detailed HPLC information is provided hereinabove.
Compound III - deoxycholic acid sodium salt
To a reaction vessel were added compound I (2 g, 5.09 mmol) and 1 N aqueous solution of sodium hydroxide (5 mL, 1 eq). The mixture was heated to 90°C for a minimum of 30 min (solubilisation observed). The solution was then allowed to cool to 30°C, at which point the solution was added to a large volume of acetonitrile (40 mL, 20V). Upon addition of the reaction mixture into the acetonitrile the product precipitated. The precipitate was separated and then taken up in water (100 mL, 50V) which was then evaporated at 90°C under vacuum to dryness (to remove all traces of organic solvent). The resulting solid was then taken up in water (100 mL, 50V) and lyophilised to provide a white solid (2.0 g, 95%), 1H NMR (300 MHz, MeOD):
6 4.02-3.95 (m, 1 H), 3.62-3.44 (m, 1 H), 2.35-2.15 (m, 1 H), 2.15-2.01 (m, 1 H), 0.95 (s, 3H), 0.73 (s, 3H), optical rotation: [a]o23 C = +45.6° (H2O, c = 1.0, literature: +46.3° - Norman Arkiv foer Kemi, 1956, 8, 331), HPLC purity 99.36%. The product had a retention time of 11.1 minutes, detailed HPLC information is provided hereinabove. The yield of each step of the synthesis starting from ethicholanolone (formula (1)) is as follows:
The overall yield starting from ethicholanolone was 7.5%.
The deoxycholic acid prepared by the process of Example 1 is a synthetic deoxycholic acid free of any human or animal-derived substance as the process of Example 1 relies on the use of non-animal derived starting materials and reagents.
Example 2
Extraction of OMVs from Neisseria meningitidis serogroup B (MenB)
Materials and Methods
Neisseria meningitidis serogroup B (MenB) cultures N. meningitidis serotype B, strain 99M that was provided by the Walter Reed Army Institute of Research (WRAIR) was cultured in a chemically defined medium described in Fu et al. (Biotechnology (N Y). 1995 Feb;13(2):170-4) and in US 5,494,808 in presence of yeast extract at 1 g/L and Hepes 1 M, at 37°C, under CO2 5%. Biomass recovery
Culture harvest was carried out using a low-speed centrifugation of the heat-treated suspension (65°C for 2 hours) in order to recover wet bacterial pellets. After centrifugation, pellets and supernatant were separated. Pellets were resuspended using centrifugation supernatant to achieve an end a volumetric concentration factor (VCF) of 5.5. dOMV extraction
Extraction was performed by mixing 1 .7X extraction buffer (either with animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC (compound (III) from Example 1 i.e. free of any human or animal-derived substance) and recovered biomass to achieve a ratio detergent/biomass of 0.5%. Table 1 - Composition of 1. 7X extraction buffer for 1 liter of buffer:
Suspensions were heated up to 56°C over 15 minutes in 2 distinct vessels.
Benzonase treatment
20 UI/mL of benzonase and 12 mM of MgCh were added to the suspension post extraction. The suspension was heated to 37°C over 30 minutes to digest residual DNA. dOMV recovery
Both suspensions from animal derived and synthetic extractions (-140 g) were centrifuged at 16000xg over
1 hour. Supernatants were recovered and pellets were discarded.
Analytics
Protein quantification was determined by Lowry assays. Protein profile was assessed by SDS-Page. SDS Page gel was prepared by charging it with 5 pg and 10 pg of proteins. Capsular polysaccharide content was determined by high pressure size exclusion chromatography (HPSEC).
Analysed stages were as follow:
Post dOMV recovery = dOMV extracts o C1 = condition 1 = extraction performed with animal derived NaDOC o C2 = condition 2 = extraction performed with synthetic NaDOC (i.e. compound (III) of Example 1 , free of any human or animal-derived substance).
Results
Fig. 1 shows protein quantification results on dOMV extracts from a Lowry Peterson assay. The protein content is similar for both conditions, i.e. animal derived NaDOC or synthetic NaDOC (i.e. compound (III) of Example 1).
Fig.2 shows that protein profiles are similar for both protein quantities and conditions of extraction (SDS- Page of dOMV extracts (5 and 10 pg)).
Fig. 3 also shows that capsular polysaccharide content is also similar for both conditions.
Conclusion
Based on the results obtained from the protein content, protein profile and capsular polysaccharide content, it can be concluded that both animal derived and synthetic NaDOC were similarly effective regarding dOMV preparation. Compound (III) is then an efficient alternative to animal derived NaDOC and presents the advantage of being devoid of the contamination risk encountered with the use of animal derived raw materials, well known in the pharmaceutical, vaccine or cosmetic industries.
Example 3
Production of a MenB vaccine OMV Drug Substance (DS)
Materials and Methods
Neisseria meningitidis serogroup B (MenB) cultures
N. meningitidis serotype B strain 99M was cultured in chemically defined medium at 30L scale, at 37°C, under CO2 5% and stopped when OD6oonm>42. Biomass recovery
Culture harvest was carried out using microfiltration 0.22pm of the heat-treated suspension (65°C for 2 hours) in order to achieve an end volumetric concentration factor (VCF) of 5.5. dOMV extraction
Extraction was performed by mixing 1 ,7X extraction buffer (see table 1 above) (either with animal derived Sodium Deoxycholate (NaDOC) or synthetic NaDOC (compound (III) from Example 1 , that is, free of any human or animal-derived substance) and recovered biomass to achieve a ratio detergent/biomass of 0.5%.
Suspensions were heated up to 56°C over 15 minutes in 2 distinct vessels.
Benzonase treatment 20 UI/mL of benzonase and 12 mM of MgCh were added to the suspension post extraction. The suspension was heated to 37°C over 30 minutes to digest residual DNA. dOMV recovery and purification dOMV were recovered and purified using the same recovery and purification steps until the DS is obtained, on one side from the suspension from animal derived extraction and on another side from the synthetic extraction. For the DS comprising the dOMV extracted using the synthetic NaDOC, the process of manufacturing of this OMV DS is free of any human or animal-derived substance and the obtained OMV DS is free of any human or animal-derived substance.
Analytics
Protein quantification was determined by Lowry assays. Protein profile was assessed by SDS-Page. Capsular polysaccharide content was determined by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).
Results
Table 2: DS characterization on both conditions
Fig. 4 shows protein quantification results on dOMV extracts from a Lowry Peterson assay at different steps of the manufacturing process of the DS: from the post benzonase treatment (post OMV extraction), through the recovery step and the purification steps, to the final DS composition. Results are shown for the DS obtained via dOMV extraction using the animal-derived NaDOC or using the synthetic NaDOC.
Conclusion
Based on the results obtained from the protein content, protein profile and capsular polysaccharide content and shown in Table 2 and Fig. 4, it can be concluded that the synthetic NaDOC has exactly the same impact on the dOMV product as the one which has animal origin. Synthetic NaDOC is therefore an efficient alternative to animal-derived NaDOC in the process for manufacturing a DS or a vaccine comprising such DS, particularly to obtain a manufacturing process free of any human or animal-derived substance.

Claims

1 . A process for preparing a compound of formula (7), comprising the step of reacting a compound of formula (6) with a reducing agent: wherein PG is a protecting group.
2. The process according to claim 1 , wherein the reduction is diastereoselective with a diastereomeric ratio (a/p) of at least 85/15 in favour of the a-isomer of the compound of formula (7):
3. The process according to claim 1 or 2, wherein the reducing agent is lithium tri-tert- butoxyaluminium hydride (LTBA) or lithium aluminium hydride (LiAIF ), and/or wherein PG is selected from an ether, silyl ether, ester, sulfonate, carbonate or carbamate protecting group.
4. The process according to any one of claims 1 to 3, further comprising the step of modifying the compound of formula (7) to form a compound of formula (9): wherein the PGs are independently as defined hereinbefore for the compound of formula (6) and (7).
5. The process according to claim 4, comprising the step of deprotecting the compound of formula (7) to provide a compound of formula (8):
(7) (8) and protecting the alcohol groups by reacting the compound of formula (8) with PG-X to form a compound of formula (9):
(8) 0) wherein the PGs are independently as defined hereinbefore for the compound of formula (7) and X is a leaving group.
The process according to claim 4 or 5, further comprising the step of reacting the compound of formula (9) with a compound of formula (b) in the presence of a coupling agent to form a compound of formula (10):
(9) (10) wherein R4 is C1-6 alkyl, and wherein the PGs are as defined hereinbefore for the compound of formula (9), optionally further comprising the step of reducing the compound of formula (10) to form a compound of formula (11) using a reducing agent:
(10) (11) wherein the PGs and R4 are as defined hereinbefore for the compounds of formula (9) and (10), optionally further comprising the step of deprotecting the protecting groups and hydrolysing the - OR4 group to provide deoxycholic acid: wherein the PGs and R4 are as defined hereinbefore for the compounds of formula (9), (10) and (11), optionally wherein the deprotection reaction is suitable to remove the protecting groups and hydrolyse the -CO2R4 group to a CO2H group in a single step.
7. The process according to claim 6, further comprising the step of converting deoxycholic acid into a pharmaceutically acceptable salt of deoxycholic acid: wherein Z+ is an organic cation, inorganic cation or metal cation, optionally wherein deoxycholic acid is converted to the sodium salt of deoxycholic acid:
8. The process according to any preceding claim, wherein the compound of formula (6) is prepared by reacting a compound of formula (5) with an oxidising agent:
(5) (6) wherein PG is as defined hereinbefore for the compounds of formula (6) and (7), optionally wherein the compound of formula (5) is prepared by reacting a compound of formula (4) with an ethyltriphenylphosphonium salt:
wherein PG is as defined hereinbefore for the compounds of formula (5), (6) and (7), optionally wherein the compound of formula (4) is prepared by reacting a compound of formula (3) with PG-X: wherein PG is as defined hereinbefore for the compounds of formula (4), (5), (6) and (7), and X is a leaving group, optionally wherein the compound of formula (3) is prepared by reacting a compound of formula (2) with an oxidising agent and a source of copper, and then hydrolysis of the imine to provide the compound of formula (3): wherein R1 is selected from C1-6 alkyl or hydrogen;
R2 and R3are selected from C1-6 alkyl or hydrogen; and n is selected from 0 or 1 , optionally wherein the compound of formula (2) is prepared by reacting a compound of formula (1) with an amino pyridine derivative of formula (a): wherein R1, R2, R3 and n are as defined hereinbefore for the compound of formula (2).
9. A process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof, wherein the process comprises the process of any preceding claim.
10. Deoxycholic acid or a pharmaceutically acceptable salt thereof obtainable by the process according to claim 9.
11. A pharmaceutical, immunogenic or cosmetic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof, prepared according to the process according to claim 9, or a pharmaceutical, immunogenic or cosmetic composition comprising deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10.
12. Use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9 in the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition, or use of deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10 in the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition.
13. The use according to claim 12 wherein the deoxycholic acid or a pharmaceutically acceptable salt thereof is used as solubilizing agent, detergent, surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial.
14. Use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9 for extracting complexes comprising at least one lipid and at least one protein, from bacteria, or use of deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10 for extracting complexes comprising at least one lipid and at least one protein, from bacteria.
15. The use according to claim 14 wherein said complexes are selected from proteoliposomes, proteasomes, or proteolipidic vesicles.
16. Use of deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9 for extracting outer membrane vesicles from Gram-negative bacteria, or the use of deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10 for extracting outer membrane vesicles from Gram-negative bacteria, optionally wherein the Gramnegative bacteria is Neisseria meningitidis serogroup B (MenB).
17. A composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10 or prepared according to the process as claimed in claim 9, and an outer membrane vesicle (OMV), optionally wherein the OMV is from Neisseria meningitidis or Neisseria meningitidis serogroup B (MenB).
18. The use of a compound of formula (1), (2), (2a), (3), (4), (4a), (5), (5a), (6), (6a), (7), (7a), (8), (9), (9a), (10), (10a), (11) or (1 1 a) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof.
19. A process for manufacturing a pharmaceutical, immunogenic or cosmetic composition comprising at least one step using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9, or using the deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10.
20. The process according to claim 19 wherein the immunogenic composition is a vaccine.
21. The process according to claim 19 or 20 wherein the deoxycholic acid or a pharmaceutically acceptable salt thereof is used as solubilizing agent, detergent, surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial.
22. A process for extracting complexes comprising at least one lipid and at least one protein, from bacteria, using the deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9 or the deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10.
23. The process according to claim 22 wherein said complexes are selected from proteoliposomes, proteasomes, or proteolipidic vesicles.
24. A process for extracting outer membrane vesicles from Gram-negative bacteria using deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9, or using the deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10.
25. A process for extracting outer membrane vesicles from Neisseria meningitidis bacteria, optionally from Neisseria meningitidis serogroup B (MenB) bacteria, using deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9, or using the deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10.
26. The process according to claim 24 or 25 comprising the steps of: a) Cultivating the bacterial cells; b) Collecting and/or concentrating the cultivated cells; c) Disrupting the outer membranes of the cultivated cells with deoxycholic acid or a pharmaceutically acceptable salt thereof prepared according to the process as claimed in claim 9, or with the deoxycholic acid or a pharmaceutically acceptable salt thereof according to claim 10, and forming outer membrane vesicles; and d) Recovering the outer membrane vesicles.
27. Use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof in the manufacturing process of a pharmaceutical, immunogenic or cosmetic composition.
28. The use according to claim 27 wherein the manufacturing process is free of any human or animal- derived substance.
29. The use according to claim 27 or 28 wherein the deoxycholic acid or a pharmaceutically acceptable salt thereof is used as solubilizing agent, detergent, surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial.
30. The use according to any one of claims 27 to 29 wherein the immunogenic composition is a vaccine.
31. Use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof for extracting complexes comprising at least one lipid and at least one protein, from bacteria.
32. The use according to claim 31 wherein the said complexes are selected from proteoliposomes, proteasomes or proteolipidic vesicles.
33. Use of a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof for extracting outer membrane vesicles from Gram-negative bacteria, optionally wherein the Gram-negative bacteria is Neisseria meningitidis or Neisseria meningitidis serogroup B (MenB).
34. A composition comprising a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof and an outer membrane vesicle (OMV).
35. The composition according to claim 34, wherein the OMV is from Neisseria meningitidis, optionally from Neisseria meningitidis serogroup B (MenB).
36. The composition according to claim 34 or 35, wherein the composition is free of any human or animal-derived substance.
37. A process for manufacturing a pharmaceutical, immunogenic or cosmetic composition comprising at least one step using a synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof.
38. The process according to claim 37 wherein the synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof is used as solubilizing agent, detergent, surfactant, reagent to precipitate a component of a composition, adjuvant or adjuvant component, component of a delivery system, cell culture medium component or bacteria culture medium component, or antimicrobial.
39. The process according to claim 37 or 38, wherein the said process is free of any human or animal- derived substance.
40. A process for extracting complexes comprising at least one lipid and at least one protein, from bacteria, using a synthetic deoxycholic acid or a pharmaceutically acceptable salt.
41. A process for extracting outer membrane vesicles from Gram-negative bacteria using a synthetic deoxycholic acid or a pharmaceutically acceptable salt, optionally wherein the Gram-negative bacteria is Neisseria meningitidis or Neisseria meningitidis serogroup B (MenB).
42. The process according to claim 40 or 41 comprising the steps of: a) Cultivating the bacterial cells b) Collecting and/or concentrating the cultivated cells c) Disrupting the outer membranes of the cultivated cells with synthetic deoxycholic acid or a pharmaceutically acceptable salt thereof and forming outer membrane vesicles d) Recovering the outer membrane vesicles.
43. Use of a compound of formula (1) in a process for preparing deoxycholic acid or a pharmaceutically acceptable salt thereof:
44. A compound of formula (2), (2a), (5), (5a), (6), (6a), (7), (7a), (9), (9a), (10), (10a), (11) or (11 a).
PCT/US2025/016340 2024-02-16 2025-02-18 Process for the preparation of deoxycholic acid Pending WO2025175304A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24158094 2024-02-16
EP24158094.3 2024-02-16

Publications (2)

Publication Number Publication Date
WO2025175304A2 true WO2025175304A2 (en) 2025-08-21
WO2025175304A3 WO2025175304A3 (en) 2025-11-06

Family

ID=89983277

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/016340 Pending WO2025175304A2 (en) 2024-02-16 2025-02-18 Process for the preparation of deoxycholic acid

Country Status (1)

Country Link
WO (1) WO2025175304A2 (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4695624A (en) 1984-05-10 1987-09-22 Merck & Co., Inc. Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and of confirming covalency
US5494808A (en) 1994-09-15 1996-02-27 Merck & Co., Inc. Defined medium OMPC fermentation process
WO2001091788A1 (en) 2000-06-02 2001-12-06 Statens Institutt For Folkehelse Outer membrane vesicle vaccine against disease caused by neisseria meningitidis serogroup a and process for the production thereof
WO2005004908A1 (en) 2003-07-15 2005-01-20 Chiron Srl Ultrafiltration for preparing outer membrane vesicles
WO2008157635A2 (en) 2007-06-19 2008-12-24 Kythera Biopharmaceuticals, Inc. Synthetic bile acid composition, method, and preparation
WO2017211820A1 (en) 2016-06-06 2017-12-14 Bionice, S.L.U. Methods for the preparation of deoxycholic acid, and intermediates useful in the preparation of deoxycholic acid
EP3284748A1 (en) 2015-04-14 2018-02-21 Noratech Pharmaceuticals, Inc. Method for preparing deoxycholic acid
WO2019024920A1 (en) 2017-08-03 2019-02-07 Medytox Inc. Methods for preparing bile acids

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002009643A2 (en) * 2000-07-27 2002-02-07 Children's Hospital & Research Center At Oakland Vaccines for broad spectrum protection against diseases caused by neisseria meningitidis
US8242294B2 (en) * 2007-06-19 2012-08-14 Kythera Biopharmaceuticals, Inc. Synthetic bile acid compositions and methods
EP3138850B8 (en) * 2011-06-16 2019-09-11 Allergan Sales, LLC Compositions containing deoxycholic acid and 10% deionized water in ethanol, directed to the purification of deoxycholic acid
WO2016202872A1 (en) * 2015-06-16 2016-12-22 Glaxosmithkline Biologicals Sa Immunogenic compositions
EP3481844B1 (en) * 2016-07-11 2024-04-17 Sage Therapeutics, Inc. C7, c12, and c16 substituted neuroactive steroids and their methods of use

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4695624A (en) 1984-05-10 1987-09-22 Merck & Co., Inc. Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and of confirming covalency
US5494808A (en) 1994-09-15 1996-02-27 Merck & Co., Inc. Defined medium OMPC fermentation process
WO2001091788A1 (en) 2000-06-02 2001-12-06 Statens Institutt For Folkehelse Outer membrane vesicle vaccine against disease caused by neisseria meningitidis serogroup a and process for the production thereof
WO2005004908A1 (en) 2003-07-15 2005-01-20 Chiron Srl Ultrafiltration for preparing outer membrane vesicles
WO2008157635A2 (en) 2007-06-19 2008-12-24 Kythera Biopharmaceuticals, Inc. Synthetic bile acid composition, method, and preparation
EP3284748A1 (en) 2015-04-14 2018-02-21 Noratech Pharmaceuticals, Inc. Method for preparing deoxycholic acid
WO2017211820A1 (en) 2016-06-06 2017-12-14 Bionice, S.L.U. Methods for the preparation of deoxycholic acid, and intermediates useful in the preparation of deoxycholic acid
WO2019024920A1 (en) 2017-08-03 2019-02-07 Medytox Inc. Methods for preparing bile acids

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
"Pharmaceutical Salts: Properties, Selection, and Use", August 2002, pages: 388
B. SCH6NECKER ET AL., ACIE, vol. 42, 2003, pages 3240 - 3244
BERGE ET AL.: "Pharmaceutically Acceptable Salts", J. PHARM. SCI., vol. 66, 1977, pages 1 - 19
FRONTIERS IN IMMUNOLOGY, vol. 5, no. 121, 2014, pages 1 - 6
FU ET AL., BIOTECHNOLOGY (N Y)., vol. 13, no. 2, February 1995 (1995-02-01), pages 170 - 4
G.J. GORSE, VACCINE, vol. 13, no. 2, 1995, pages 209 - 214
HELTING ET AL., ACTA PATHOL MICROBIOL SCAND C., vol. 89, no. 2, April 1981 (1981-04-01), pages 69 - 78
J. ORG CHEM, vol. 72, 2007, pages 9298 - 9307
J.S. WILKHU ET AL., J DRUG TARGET, vol. 21, no. 3, 2013, pages 291 - 299
L. KURTIB. CZAKO: "Strategic Applications of Named Reactions in Organic Synthesis", 2005, ELSEVIER
NORMAN ARKIV FOER KEMI, vol. 8, 1956, pages 331
P.S. BARAN ET AL., JACS, vol. 137, 2015, pages 13776 - 13779
Q.P. LEI ET AL., DEVELOPMENTS IN BIOLOGICALS, vol. 103, 2000, pages 259 - 264
R. MCKENZIE ET AL., VACCINE, vol. 24, no. 18, 2006, pages 3735 - 3745
S. D. OFFEI ET AL., STEROIDS, vol. 186, 2022, pages 109088
T. GREENP. WUTS: "Protective Groups in Organic Synthesis", 1999, JOHN WILEY AND SONS
T. MU ET AL., NATURE COMMUNICATIONS, vol. 11, 2020, pages 4371
V.V.H. LE ET AL., BMC MICROBIOL., vol. 20, no. 1, 2020, pages 5
W.G. LAVER ET AL., POSTGRAD MED J., vol. 52, no. 608, 1976, pages 373 - 8

Also Published As

Publication number Publication date
WO2025175304A3 (en) 2025-11-06

Similar Documents

Publication Publication Date Title
EP1189915B1 (en) Process for the production of azithromycin
SA99191247B1 (en) Modern aromatic amides and a method for their preparation and drug use
Udodong et al. n-pentenyl glycosides in the efficient assembly of the blood group substance B tetrasaccharide
CN102219817A (en) Method for carrying out carbalkoxylation acylation on fluorouracil compound with active coupling agent
CN113831316A (en) 1-O-alkyl genipin and preparation method and application thereof
WO2025175304A2 (en) Process for the preparation of deoxycholic acid
CN114478339B (en) Preparation method of xanthate collector
EP0882733B1 (en) Acylating agent and a method of producing thereof
SE447118B (en) 4 &#34;-DEOXI-4&#34; ACYLAMIDO DERIVATIVES OF OLEANDOMYCINES, ERYTHROMYCINES AND ERYTHROMYCINE CARBONATE
JP6526692B2 (en) Novel synthetic oligomers of Neisseria meningitidis serotype X and methods for their preparation
Yang et al. Rapid assembly of phosphate-bridged tetra-mannose by ionic liquid-supported oligosaccharide synthesis
CN108892740B (en) Synthesis method of 3, 6-branched glucan hexaose
CN113372375B (en) Preparation method of temsirolimus intermediate
WO2022034429A1 (en) PROCESS OF SYNTHESIS OF β-6&#39;SULFOQUINOVOSYL DIACYLGLYCEROLS
CN114456217A (en) Synthetic method of glycal compound
CN113683539B (en) Synthesis method of polyketone intermediate
CA2319833A1 (en) Glycosidation of 4,5-epoxymorphinan-6-ols
EP0222172A2 (en) N-Glycolylneuraminic acid derivative
CA2237076A1 (en) Ursodeoxycholic acid derivatives and methods for producing them
CN109400666B (en) Preparation method of diosgenin ether
JP4813838B2 (en) O-linked sugar amino acid derivative having core 6 type structure and method for producing the same
SU457696A1 (en) Method for preparing glycol mono-tosylate vinyl ethers
EP1709057A1 (en) Synthesis of spongosine
JP3806456B2 (en) Cancer cell metastasis inhibitor 6- (trichloroacetamide) -4,5-dihydroxypiperidine-3-carboxylic acid and 6-guanidino-4,5-dihydroxypiperidine-3-carboxylic acid and related substances
JPS61172867A (en) Analogs of the non-reducing subunit of lipid A