WO2024251966A1 - Nicotinamide mononucléotide cristallin réduit (nmnh) et son procédé - Google Patents

Nicotinamide mononucléotide cristallin réduit (nmnh) et son procédé Download PDF

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Publication number
WO2024251966A1
WO2024251966A1 PCT/EP2024/065769 EP2024065769W WO2024251966A1 WO 2024251966 A1 WO2024251966 A1 WO 2024251966A1 EP 2024065769 W EP2024065769 W EP 2024065769W WO 2024251966 A1 WO2024251966 A1 WO 2024251966A1
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Prior art keywords
compound
nmnh
crystalline
formula
iii
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Ramani MALKANNAGARI
Raghava Rao GUNDAPUNENI
Frode BOHAN
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Bohan & Co AS
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Bohan & Co AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/048—Pyridine radicals

Definitions

  • NMNH reduced nicotinamide mononucleotide
  • NMNH reduced nicotinamide mononucleotide
  • BACKGROUND OF THE INVENTION Nicotinamide adenine dinucleotide (NAD+) is essential to living organisms since it participates in various biological reactions and regulates key biological processes, such as metabolism and DNA repair. Nicotinamide adenine dinucleotide (NAD+) homeostasis is constantly compromised due to degradation by NAD+ -dependent enzymes which lead to various age related diseases.
  • NMN nicotinamide mononucleotide
  • NAD+ replenishment by supplementation with the NAD+ precursors nicotinamide mononucleotide (NMN) is thought to alleviate this imbalance.
  • Recent studies have revealed that reduced nicotinamide mononucleotide (NMNH) is a potent NAD precursor than NMN both in vitro and in vivo.
  • NMNH increases cellular NADH levels, suppresses glycolysis and TCA cycle, as well as cell growth.
  • the researchers worldwide have focused on NMNH and its production on large scale such that NMNH would be available at lower prices than the precursors currently available.
  • NMNH chemically known as ((2R,3S,4R)-5-(3-carbamoylpyridin-1(4H)-yl)-3,4- dihydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate is the reduced form of NMN with the molecular formula of C 11 H 17 N 2 O 8 P, is stable under alkaline pH and low temperature conditions, but is unstable at neutral pH in solution.
  • NMN-H Given the therapeutic benefits as nutraceuticals associated with NMN-H, the present inventors envisaged the need in the art to provide novel crystalline form of NMN-H and to the process for synthesis thereof which is efficient, industrially scalable and provides NMNH in high yield and purity.
  • NMNH reduced nicotinamide mononucleotide
  • the present invention provides a process for synthesis of crystalline form of reduced nicotinamide mononucleotide (NMNH) of Formula I (I) from the compound of general Formula (II) or (IIa); (II) or (IIa) wherein R is selected from hydrogen or –P(O)R1R2; R1 and R2 independently represent OH and O-respectively; and X- is halo, or triflate. comprising; i. Ketalizing the compound of Formula II, wherein R is hydrogen; X- is halo or triflate to a compound of Formula III
  • R3 and R4 are alkyl, X- is halo or triflate ;
  • Phosphorylating compound of Formula (III) with PO(OR 5 )Cl to obtain the compound of Formula (IV) wherein R3, R4, R5, R6 and R7 are independently alkyl, X- is halo or triflate;
  • the present invention provides a composition comprising the crystalline compound of reduced nicotinamide mononucleotide of Formula (I) with suitable excipients.
  • FIGURES Figure 1 depict the chromatogram of as prepared NMNH.
  • Figure 2 depict the Masss pectra of as prepared NMNH
  • Figure 3 depict the Powder X-RD of NMN
  • Figure 4 depict the Powder X-RD of NMNH DETAILED DESCRIPTION OF THE INVENTION:
  • the present invention will now be described in detail in its preferred and optional embodiments so that various aspects of the invention will be fully understood without limiting the scope of the invention.
  • NMNH reduced nicotinamide mononucleot
  • the present invention discloses a process for synthesis of crystalline form of reduced nicotinamide mononucleotide (NMNH)of Formula I from the compound of general Formula (II) or (IIa) ; (II) or (IIa) wherein R is selected from hydrogen or –P(O)R1R2; R1 and R2 independently represent OH and O-respectively; and X- is halo or triflate. comprising; i. Ketalizing the compound of Formula II, wherein R is hydrogen; X- is halo or triflate; to a compound of Formula III wherein R3 and R4 are alkyl, X- is halo or triflate; ii. Phosphorylating compound of Formula (III) with PO(OR 5 )Cl to obtain the compound of Formula (IV)
  • R3, R4, R5, R6 and R7 are independently alkyl, X- is halo or triflate; iii. Reducing compound (IV) with dithionite under ice-cold conditions to obtain compound V; wherein R3, R4, R6 and R7 are independently alkyl; and iv. Deprotecting the compound of Formula (V) and purifying in methanol to obtain reduced nicotinamide mononucleotide of formula (I). OR i.
  • the present invention relates to a process for synthesis of crystalline form of reduced nicotinamide mononucleotide (NMNH) of Formula I from the compound of general Formula (II); Wherein R is selected from hydrogen or –P(O)R1R2; R1 and R2 independently represent OH and O- respectively; and X- is halo or triflate.
  • R is selected from hydrogen or –P(O)R1R2; R1 and R2 independently represent OH and O- respectively; and X- is halo or triflate.
  • R3 and R4 are independently alkyl, X- is halo or triflate; ii. Phosphorylating compound of Formula (III) with PO(OR 5 )Cl to obtain the compound of Formula (IV) wherein R3, R4, R5, R6 and R7 are independently alkyl; iii. Reducing compound (IV) with dithionite under ice-cold conditions to obtain compound V; wherein R3, R4, R6 and R7 are independently alkyl; and iv. Deprotecting the compound of Formula (V) and purifying to obtain reduced nicotinamide mononucleotide of formula (I).
  • the X- anion in the compound of Formula (II), (IIa), (III) and (IV) is preferably chloro. Accordingly, in the step (i) of the process of the present invention as shown above, the ketalization of compound (II) to compound (III) is carried out using the ketalizing agent selected from cyclic or acyclic ketones such as 2,2dimethoxy propane (2,2 DMP), 2,2 diethoxy propane, acetone, and the like.
  • the ketalization process step (i) is catalyzed by acid, wherein the acid catalyst can be an inorganic acid catalyst such as sulfuric acid, hydrochloric acid, phosphoric acid, oleum and the like alone or mixtures thereof.
  • the acid catalyst can be an organic acid catalyst, for example, p-toluenesulfonic acid, methylsulfonic acid, trifluoromethylsulfonic acid, and the like alone or mixtures thereof.
  • the acid catalyst is a mixture of sulfuric acid and oleum.
  • the solvent can be any suitable solvent such as acetonitrile, dichloromethane, acetone, dimethylformamide, dimethylsulfoxide, and the like alone or mixture thereof.
  • the solvent is acetonitrile.
  • the step of ketalization is performed at a suitable temperature ranging between 0 °C to 25°C
  • the phosphorylation of compound (III) to compound (IV) is carried out using phosphorylating agent selected from suitable phosphorylating agent, preferably the phosphorylating agent is di-tert-butylchlorophosphate.
  • the phosphorylation is carried out in presence of base selected from organic or inorganic base and in suitable solvent selected from polar or non-polar, protic or aprotic solvent alone or mixtures thereof.
  • the reaction is carried out at a suitable temperature ranging between 0°C to 25°C
  • the step (iii) of the process which comprises reduction of the salt of compound (IV) to the base (compound of formula V) is carried out in presence of reducing agent such as sodium dithionite at alkaline pH using a suitable base and under ice- cold conditions.
  • the solvent is selected from water, acetates, ethers, alcohols, hydrocarbons and the like alone or mixtures thereof.
  • the step (iv) of deprotection of compound (V) to NMNH is conducted preferably in methanol in the presence of HCl or formic acid or TFA catalyst.
  • the reduced nicotinamide mononucleotide (NMNH) obtained by the process of the present invention is further isolated and purified from the solvent methanol.
  • the present invention relate to a process for synthesis of crystalline NMNH (I) comprising; i. Ketalizing nicotinamide riboside chloride (NRCl) (II) with 2,2DMP in presence of H2SO4 and oleum in acetonitrile to obtain the intermediate compound (III); ii. Phosphorylating the compound (III) of step (i) with di-tert- butylchlorophosphate in presence of base and solvent to obtain the intermediate compound (IV); iii.
  • the present invention discloses a process for synthesis of crystalline form of reduced nicotinamide mononucleotide (NMN-H) of formula (I); comprising; i. Adding portion wise sodium dithionite as reducing agnet in alkaline pH and under ice –cold conditions to the compound of Formula (IIa) wherein R is–P(O)R1R2 R1 and R2 independently represent OH and O-respectively; and X- is halo; ii. Concentrating the mixture of step (i) to half its volume by evaporation; iii. Cooling the temperature to 0 o C and precipitating the mixture from the solvent; iv.
  • step (iii) Filtering the precipitate of step (iii), concentrating the filtrate followed by dissolving it in DI water and solvent at 0 o C until precipitation of salts from the filtrate; and v. Concentrating the filtrate and crystallizing to obtain desired product.
  • base selected from suitable bases which include inorganic bases such as alkali and alkaline earth metal bases, e.g., those containing metallic cations such as sodium, potassium, magnesium, calcium and the like alone or mixtures thereof, organic base such as ethylamine, pyridine and the like followed by portion wise addition of the reducing agent such as sodium dithionite and allowing to stir at the same temperature. This is followed by concentrating the reaction mixture to half its volume and precipitating the mass from the mixture of solvents.
  • suitable bases include inorganic bases such as alkali and alkaline earth metal bases, e.g., those containing metallic cations such as sodium, potassium, magnesium, calcium and the like alone or mixtures thereof, organic base such as ethylamine, pyridine and the like followed by portion wise addition of the reducing agent such as sodium dithionite and allowing to stir at the same temperature. This is followed by concentrating the reaction mixture to half its volume and precipitating the mass from the mixture of solvents.
  • inorganic bases such as
  • the precipitate is further washed, filtered and the filtrate is concentrated under vacuum to obtain crystalline NMNH of Formula (I).
  • the solvent used in the process can be any suitable solvent selected from polar or non-polar, protic or aprotic solvent such as water, lower alcohols, ketone, ethers, acetates and such like alone or mixtures thereof.
  • the present invention relates to a process for synthesis of crystalline reduced nicotinamide mononucleotide (NMNH) of formula (I) from nicotinamide mononucleotide (NMN) of formula (Ia) comprising; i.
  • NMN nicotinamide mononucleotide
  • IIa nicotinamide mononucleotide
  • step (i) Concentrating the mixture of step (i) to half its volume by evaporation; iii. Cooling the temperature to 0 o C and precipitating the mixture from the solvent; iv. Filtering the precipitate of step (iii) and concentrating the filtrate followed by dissolving it in DI water and then addition of the solvent at 0 o C until precipitation of salts from the filtrate; and v. Concentrating the filtrate under reduced pressure followed by purification in methanol to obtain NMNH.
  • NPN nicotinamide mononucleotide
  • the process is depicted in Scheme 2 below:
  • the crystalline reduced nicotinamide mononucleotide (NMNH) of formula I obtained by the process of the present invention is further characterized by chromatogram, Mass spectra, as shown in the figures 2 and 3.
  • the process of the present invention avoids the use of costly chromatographic techniques used in the art, which makes the present process cost effective and industrially viable.
  • the crystalline reduced nicotinamide mononucleotide (NMNH) of formula I is stable at 2-8oC for a period of 12-24months.
  • the pharmaceutical composition and/or nutraceutical/dietary supplement compositions comprises the crystalline NMNH (I) prepared by the process of the present invention is provided herein.
  • the pharmaceutical/nutraceutical/ dietary supplement composition may be formulated using suitable formulating excipients, adjuvants in appropriate amounts.
  • the nutraceutical/dietary supplement composition may be in the form of powder, granules, pellets, syrups, suspensions and such suitable forms which may be taken along with food.
  • the pharmaceutical composition may conveniently be provided in unit dosage form and may be prepared by any methods well known in the art. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • Example 1 Synthesis of crystalline reduced Nicotinamide Mononucleotide (Formula I) from Nicotinamide Riboside Chloride (NRCl) (Formula II)
  • Example 1a Preparation of compound (III) from Nicotinamide Riboside Chloride (NRCl) Formula (II)
  • H2SO4 4.3 mL, 80 mmol
  • Example 1b Preparation of compound (IV) through the phosphorylation of Formula III Compound-III (15g, 50.79 mmol) was dissolved in 100 mL DCM at 0 o C followed by addition of Et3N (14.15 mL, 101.59 mmol) and DMAP (1.24g, 10.15 mmol).
  • Example 1c Preparation of compound (V) by the reduction of IV
  • the compound-IV 14g, 28.71 mmol
  • EtOAc 200 mL
  • 65 mL sat.NaHCO3 solution were added, followed by addition of solid sodium dithionite (26.24g, 150.77mmol) and 35 mL of water at stirring and at room temperature.
  • the biphasic reaction mixture was stirred at room temperature for 6h, and then EtOAc (200 mL) was added.
  • the organic phase was separated, and evaporated under reduced pressure to give the reduced form of compound V.
  • Example 1d Preparation of NMNH (I) from compound of Formula V.
  • Example 2 Preparation of Crystalline Reduced ((2R,3S,4R)-5-(3- carbamoylpyridin-1(4H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl di hydrogen phosphate (NMNH) from Nicotinamide Mononucleotide (NMN) (Formula IIa) To the stirred solution of NMN (8.15g, 1 equiv, 24.38 mmol) in 100 mL deionized water at 0 o C was added saturated NaHCO 3 solution (200 mL) in a single portion.
  • NMN Nicotinamide Mononucleotide
  • Example 3 Characterization of Crystalline NMNH and NMN Powder X-ray diffraction studies: Powder X-ray diffraction studies were carried out in Bragg–Brentano (reflection) geometry on a Bruker D8 Advance Davinci diffractometer and D2 Phaser diffractometers equipped with a Cu X-ray source (1.5418 ⁇ ) and an LYNXEYE- XET high-resolution position-sensitive detectors. The samples were placed into the plate sample holders and rotated at a rate of 15 rpm during the data acquisition.
  • Powder X-RD of NMN 7.559 o , 11.434 o , 12.556 o , 15.947 o , 18.065 o , 18.870 o , 19.039 o , 20.027 o , 20.707 o, 20.914 o , 21.717 o , 22.962 o , 23.238 o , 25.387 o , 25.612 o , 26.491 o , 29.646 o , 30.244 o , 30.898 o ⁇ 2 ⁇ Powder X-RD of NMNH 19.112 o , 23.024 o , 25.340 o , 31.986 o , 33.569 o , 47.050 o ⁇ 2 ⁇ .

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  • Genetics & Genomics (AREA)
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  • Saccharide Compounds (AREA)
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Abstract

La présente invention concerne une nouvelle forme cristalline de nicotinamide mononucléotide (NMNH) réduit de formule (I) et son procédé de préparation.
PCT/EP2024/065769 2023-06-07 2024-06-07 Nicotinamide mononucléotide cristallin réduit (nmnh) et son procédé Ceased WO2024251966A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017059249A1 (fr) 2015-10-02 2017-04-06 Metrobiotech, Llc Formes cristallines de bêta-nicotinamide mononucléotide
WO2021098725A1 (fr) * 2019-11-19 2021-05-27 Tsinghua University Procédé de synthèse d'un dérivé du nmn et applications médicales du nmn et de son dérivé
WO2021214299A1 (fr) * 2020-04-24 2021-10-28 Nuvamid Sa Dérivés de nicotinamide mononucléotide et de nicotinamide riboside et leur utilisation dans le traitement d'infections virales et de complications respiratoires, provoquées en particulier par le virus de la grippe ou le coronavirus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017059249A1 (fr) 2015-10-02 2017-04-06 Metrobiotech, Llc Formes cristallines de bêta-nicotinamide mononucléotide
WO2021098725A1 (fr) * 2019-11-19 2021-05-27 Tsinghua University Procédé de synthèse d'un dérivé du nmn et applications médicales du nmn et de son dérivé
WO2021214299A1 (fr) * 2020-04-24 2021-10-28 Nuvamid Sa Dérivés de nicotinamide mononucléotide et de nicotinamide riboside et leur utilisation dans le traitement d'infections virales et de complications respiratoires, provoquées en particulier par le virus de la grippe ou le coronavirus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Proton Magnetic Resonance Study of the intramolecular Association and Confirmation of the alpha- and beta-Nicotine Mononucleotides and Nucleosides", BIOCHEMISTRY, vol. 15, no. 18, pages 3981 - 3983

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