ES2798435T3 - Method for preparing nicotinoyl ribosides and nicotinamide beta-riboside - Google Patents

Abstract

A method for preparing nicotinamide riboside of Formula I: ** (See formula) ** wherein R1 is amino, comprising the steps of: (i) mixing (a) substituted tetraacyl or acyl ribofuranose and (b) a compound of formula II: ** (See formula) ** where, R2 is alkyloxy, -OCH2CH2OH or aryloxy; to form a compound of formula III: ** (See formula) ** wherein, R2 is alkyloxy, -OCH2CH2OH, or aryloxy, and R3, R4, and R5 are each acyl; and (ii) mixing the compound of formula III with a solution of ammonia to form nicotinamide riboside.

Description

DESCRIPTION

Method for preparing nicotinoyl ribosides and nicotinamide beta-riboside

Field of the invention

The invention relates to a method for preparing nicotinoyl ribosides and nicotinamide ribosides. In some embodiments, the description refers to pharmaceutical compositions and nutritional supplements that contain a nicotinoyl riboside. In some embodiments, the disclosure relates to methods of using nicotinoyl ribosides and nicotinamide riboside derivatives that promote increased intracellular levels of nicotinamide adenine dinucleotide (NAD +) in cells and tissues to enhance cell and tissue survival.

Background of the invention

Nicotinamide adenine dinucleotide (NAD +) is a natural coenzyme that functions as an intermediate in cellular oxidation and reduction reactions, as well as a substrate for ADP-ribosyltransferase. Altering intracellular levels of NAD + can improve the health of a cell, but the introduction of compounds that enter the metabolic pathways of NAD + can also be toxic to cells. For example, benzamide riboside (BAR) is a well known antitumor agent. BR is a prodrug that can be phosphorylated to its 5'-monophosphate and then converted to its active metabolite benzamide adenine dinucleotide (BAD). This metabolite is an active analog of NAD + and an inhibitor of inosine 5'-monophosphate dehydrogenase (IMPHD). IMPDH is linked to malignant transformations. BAR shows selective sensitivity to the central nervous system and to leukemic cell lines. However, BAD also inhibits other dehydrogenases, such as malate dehydrogenase and glutamic acid dehydrogenase which can cause adverse effects if used as a therapeutic. Therefore, a need exists to identify compositions that are capable of improving the health of diseased or damaged cells preferably by altering intracellular levels of NAD + and that do not have adverse effects if the compositions are administered therapeutically.

Summary of the invention

The invention relates to a method for preparing nicotinoyl ribosides and nicotinamide ribosides according to present claims 1 to 7. In some embodiments, the description refers to pharmaceutical compositions and nutritional supplements containing a nicotinoyl riboside. In other embodiments, the disclosure relates to methods of using nicotinoyl ribosides and nicotinamide riboside derivatives that promote increased intracellular levels of nicotinamide adenine dinucleotide (NAD +) in cells and tissues to enhance cell and tissue survival.

In some embodiments, the invention relates to a method of preparing a compound having Formula I:

Fórmula I

Formula I

wherein, R1 is amino, comprising: i) mixing a) substituted tetraacyl or acyl ribofuranose and b) a compound having formula II:

Fórmula II

Formula II

wherein R2 is alkyloxy, -OCH ² CH ² OH, aryloxy; to form a compound of formula III:

Fórmula III

Formula III

wherein R2 is alkyloxy, -OCH ² CH ² OH, aryloxy, and R3, R4, and R5 are each acyl; and ii) mixing the compound of formula III with an ammonia solution to form a compound of formula I. In additional embodiments, the conditions for preparing compound I from compound III are in a halogenated alkyl alcohol solvent at a lower temperature. at room temperature. In other embodiments, said solvent is trifluoromethanol.

In further embodiments, the description refers to a method of preparing a compound having the Formula

wherein R1 is amine, substituted amine, alkylamine, substituted alkylamine, dialkylamine, substituted dialkylamine, alkyloxy or substituted alkyloxy,

comprising: i) mixing a) substituted tetraacyl or acyl ribofuranose, preferably tetraacetyl ribofuranose, and b) a compound having formula II:

Fórmula II

Formula II

wherein R2 is alkyloxy, substituted alkyloxy, aryloxy, substituted aryloxy, alkylthiol, substituted alkylthiol, arylthiol, or substituted arylthiol under conditions such that a compound of formula III is formed:

Fórmula III

Formula III

wherein R2 is alkyloxy, substituted alkyloxy, aryloxy, substituted aryloxy, alkylthiol, substituted alkylthiol, arylthiol, or substituted arylthiol, and R3, R4, and R5 are the same or different and, in each case independently, acyl or substituted acyl; and ii) mixing the compound of formula III with ammonia, ammonia substituted alkylamine, substituted alkylamine, dialkylamine, substituted dialkylamine, alkoxide or substituted alkoxide under conditions such that a compound of formula I is formed.

In some embodiments, the invention relates to a method of preparing a compound having Formula I:

Fórmula I

Formula I

wherein, R is NH ² , alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, alkyloxy, and substituted alkyloxy, comprising: i) mixing tetra-acyl ribofuranose, preferably tetraacetyl ribofuranose, and the compound of alkyl or aryl pyridine-3-carboxylate ester or thioester under conditions such that a compound of formula II is formed:

Fórmula II

Formula II

where R2 is -O-alkyl, -O-aryl, -S-alkyl or -S-aryl and, R3, R4 and R5 are the same or different and, in each case, independently acyl and iii) mixing the compound of formula II with an alkylamine, substituted alkylamine, dialkylamine, substituted dialkylamine, alkoxide, and substituted alkoxide under conditions such that a compound of formula I is formed.

In some embodiments, the description refers to the preparation of alkyl 1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate, preferably 1 - [3,4-diacetyloxy ] -5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-alkyl carboxylate, which comprises mixing a composition consisting essentially of 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose , alkylpyridine-3-carboxylate and trimethylsilyl trifluoromethanesulfonate under conditions such that alkyl 1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine 3-carboxylate is formed.

In some embodiments, the description refers to a method of preparing N-alkyl or N, N-dialkyl 1- [3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] pyridine-5-carboxamide comprising mixing alkyl 1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate with a primary or secondary amine under conditions such that 1- [3,4-dihydroxy- N-alkyl or N, N-dialkyl 5- (hydroxymethyl) oxolan-2-yl] pyridine-5-carboxamide.

In some embodiments, the description refers to a method of preparing O-Alkyl2 1- [3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] pyridine-5-carboxylate which comprises mixing 1- [3, 4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] pyridine-5-carboxylate of O-Alkyl1 and sodium oxide and alkyl2 under conditions such that 1- [3,4-dihydroxy-5- (hydroxymethyl) is formed O-Alkyl2 oxolan-2-yl] pyridine-5-carboxylate.

Brief description of the figures

Figure 1 shows a scheme of preferred methods for preparing preferred compositions.

Figure 2 shows data on improved cell survival when grown in medium supplemented with nicotinamide riboside (NAR) as provided in Example 7.

Figure 3 shows data on increased intracellular levels of nicotinamide adenine dinucleotide (NAD +) when embryonic stem cells are exposed to a culture containing NAR, as provided in Example 6.

Figure 4 shows data on the effects of NAR on cell survival as provided in Example 8.

Figure 5 illustrates the preparation of N-alkyl nicotinamide ribosides.

Figure 6 illustrates kinetically (-20 ° C) or thermodynamically (4 ° C) controlled reactions of O-alkyl nicotinate ribosides with alkylamines.

Figure 7 shows the mammalian NAD + biosynthetic pathway. Abbreviations: NA, nicotinic acid; Npt, nicotinic acid phosphoribosyltransferase; NaMN, nicotinic acid mononucleotide; Nmnat, nicotinamide mononucleotide adenylyltransferase; NAAD, nicotinic acid adenine dinucleotide; NAM, nicotinamide; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; Nrk, nicotinamide riboside kinase; ART, ADP-ribosyl transferase; PARP, poly-ADP-polymerases; Nampt, nicotinamide phosphoribosyltransferase, also known as pre-B cell colony enhancement factor (PBEF).

Figure 8 demonstrates the reduction in statin toxicity (lovastatin) for mouse embryonic stem cells treated with nicotinamide riboside (500 pM) for periods of 2, 4 and 7 days. Statin-treated controls are indicated by minus symbols. Bars represent statin concentration leading to a 50% decrease in cell number or the MTT assay, which measures cell viability. The leftmost two columns for each day are measured by cell count, the right two columns are measured by MTT. As shown, NR treatment significantly reduces statin toxicity on cell viability as determined by an increase in the amount of statin required to kill cells.

Figure 9 shows a bar diagram of the content of NAD + (pmoL) in ES cells after being treated with different compounds.

Figure 10 shows the percentage of cell death after a 48 hour treatment with lovastatin (800 nM and 5 gM). Mouse ES cells were treated in the presence of nicotinate riboside derivatives NAR (500 gM), OENR ⁽¹ mM), TAENR (1 mM). Abbreviations: NAR: nicotinic acid riboside, OENR: O-ethyl nicotinamide riboside, TAENR: tri-O-acetyl O'-ethyl nicotinamide riboside. Control cells were not treated with a derivative. The percentage of cell death is determined by the percentage of total viable cells in the statin treated wells versus the untreated (no statin) controls. The effect of each additive on cell death is calculated in a similar way, compared to a control without statin in which the nicotinate derivative of riboside is also present. Cell counts in untreated controls (without statins) for each experimental group were similar, indicating a lack of toxicity of the compounds on cells.

Figure 11 shows the chemical structure of the compounds tested in FIG. 10.

Figure 12 illustrates data from the cell proliferation assay with DMNR (1 mM) and ANR (1 mM) treatment. DMNR: N-dimethyl nicotinamide riboside, ANR: N-allyl nicotinamide riboside.

Figure 13 shows chemical structures of the compounds tested in FIG. 12.

Figure 14 shows 1H-NMR of nicotinamide riboside

Figure 15 shows 1H-NMR of O-ethyl nicotinate riboside.

Figure 16 shows 1H-NMR of N-methyl nicotinamide riboside.

Figure 17 shows 1H-NMR of N-ethyl nicotinamide riboside.

Figure 18 shows 1H-NMR of N-allyl-nicotinamide-riboside.

Figure 19 shows 1H-NMR of N-ethanol nicotinamide riboside.

Figure 20 shows 1H-NMR of dimethyl nicotinamide riboside.

Detailed description of the invention

The invention relates to a method for preparing nicotinoyl ribosides and nicotinamide ribosides according to present claims 1 to 7. In some embodiments, the description refers to pharmaceutical compositions and nutritional supplements containing a nicotinoyl riboside. In further embodiments, the disclosure relates to methods of using nicotinoyl ribosides and nicotinamide riboside derivatives that promote increased intracellular levels of nicotinamide adenine dinucleotide (NAD +) in cells and tissues to enhance cell and tissue survival.

A "nicotinoyl riboside" compound means a substituted or unsubstituted compound of the following formula:

The "salts" of nicotinoyl ribonucleotides refer to salts that make up the composition that includes

specifically the riboside salt of nicotinoyl having the formula p

and Z is a counter ion, including chloride, bromide, iodide, alkoxide, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, fumarate, citrate, tartrate, ascorbate, cinnamate, mandeloate and diphenylacetate).

As used herein, the term "nicotinoyl riboside component" refers to the part of a composition that contains all nicotinoyl riboside molecules in a given composition, including all conformational and stereomeric forms. In preferred embodiments, a given compound (eg, designated by a structure) constitutes a large percentage (eg, by number of molecules and / or by weight) of the nicotinoyl riboside component. For example, a given nicotinoyl riboside molecule may be present in an aqueous composition at a level where 70% of all nicotinoyl riboside molecules are from that given compound, while the majority of the composition itself is composed of of water.

"Acyl" means a -C (= O) alkyl or -C (= O) aryl group.

"Alkyl" means a straight or branched chain unsaturated or saturated non-cyclic or cyclic aliphatic hydrocarbon containing from 1 to 10 carbon atoms, while the term "lower alkyl" has the same meaning as alkyl, but contains from 1 to 6 carbons atoms. The term "higher alkyl" has the same meaning as alkyl but contains from 2 to 10 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-sepyl, n-octyl, n-nonyl, and the like; while the saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls are also referred to herein as "homocycles" or "homocyclic rings". Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as "alkenyl" or "alkynyl", respectively). Representative straight and branched chain alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3 -dimethyl-2-butenyl, and the like; while representative straight and branched chain alkynyl include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"Alkylamino" and "dialkylamino" mean one or two alkyl moieties linked through a nitrogen bridge (ie, -N-alkyl) such as methylamino, ethylamino, dimethylamino, diethylamino, and the like.

"Alkylthiol" means an alkyl moiety attached through a sulfur bridge (ie, -S-alkyl).

"Alkyloxy" means an alkyl moiety attached through an oxygen bridge (ie, -O-alkyl) such as methoxy, ethoxy, and the like.

"Alkoxide" means an alkyl moiety attached to a negatively charged oxygen atom (ie, -Oalkyl) such as methoxide or ethoxide.

In the context of a certain embodiment, an "amine" means -NH ² or "ammonia" the NH ³ gas.

"Aryl" means an aromatic carbocyclic moiety such as phenyl or naphthyl.

"Aryloxy" means an aryl moiety attached through an oxygen bridge (ie, -O-aryl).

"Arylthio" means an alkyl moiety attached through a sulfur bridge (ie, -S-aryl).

"Aminooxy" means an amino moiety attached through an oxygen bridge (ie, -O-NH ² ).

"Hydroxylamino" means a hydroxyl moiety attached through an amine bridge (ie, -NH-OH).

"O-alkyloxyamino" means an alkylamino moiety attached through an amine bridge (ie, -NH-O-alkyl). "N-alkylaminooxy" means an alkylamino moiety attached through an oxygen bridge (ie, -O-NH-alkyl). "Hydrazino" means an amino residue attached through a nitrogen bridge (ie, -NH-NH ² ) depending on the context.

"Alkylhydrazino" means an alkyl moiety attached through a hydrazine bridge (ie, -NH-NH-alkyl).

"Cells" means the structural unit of an organism that consists of one or more nuclei, cytoplasms, and various organelles, all surrounded by a semipermeable cell membrane.

"Growth media" are compositions used to grow microorganisms or cells in culture. There are different types of media for growing different types of cells. The biggest difference in growth media is found between those used to grow cells in culture (cell culture uses specific cell types derived from plants or animals) and those used to grow microorganisms (usually bacteria or yeast). These differences arise due to the fact that cells derived from whole organisms and cultured in culture are often unable to grow without the provision of certain requirements, such as hormones or growth factors that generally occur in vivo. In the case of animal cells, these requirements are often provided by the addition of blood serum to the medium. These media are often red or pink due to the inclusion of pH indicators. The growth media for embryonic stem cells preferably contain a minimal essential medium, i.e. Eagle: amino acids, salts (ferric nitrate nonahydrate, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate), vitamins (ascorbic acid, folic acid, nicotinamide, riboflavin, B-12) or Dulbecco: additionally iron, glucose; amino acids no essential oils, sodium pyruvate, p-mercaptoethanol, L-glutamine, fetal bovine serum and leukemia inhibitory factor (LIF). In the case of microorganisms, there are no such limitations as they are often single cell organisms. Another important difference is that animal cells in culture often grow on a flat surface to which they are attached, and the medium is provided in liquid form, which covers the cells. Bacteria such as Escherichia coli (E. coli, the most commonly used microbe in laboratories) can be cultured on solid media or liquid media, the liquid nutrient medium is commonly called nutrient broth. The preferred growth media for the microorganisms are nutrient broth or Luria-Bertani medium (LB medium). Bacteria grown in liquid cultures often form colloidal suspensions. When agar (a substance that turns into a gel) is added to a liquid medium, it can be poured into Petri dishes where it will solidify (called agar plates) and provide a solid medium on which microbes can grow.

"Homocycle" (also referred to herein as "homocyclic ring") means a saturated or unsaturated (but non-aromatic) carbocyclic ring containing from 3 to 7 carbon atoms, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene, and the like.

"Isomers" means any two or more substances that are composed of the same elements in the same proportions but differ in the three-dimensional arrangement of atoms that include enantiomeric (ie, mirror images) and diastereomeric isomers.

"Methylene" means -CH ² -.

The term "derivative", when used in relation to a chemical compound, refers to a similar structure which, upon application, for example administration to a subject, is capable of providing, directly or indirectly, the function of said chemical compound described as having. In the context of certain embodiments, it is understood that a "nicotinamide riboside derivative (NAR)" does not include the NAR compound.

As used herein, the terms "purified isomer" and "purified isomer composition" are intended to indicate a composition (eg, derived from a racemic mixture or synthesized de novo) in which an isomer has been enriched (eg, alpha isomer) relative to the other (e.g. beta isomer), and more preferably, wherein the other isomer represents less than 10%, and more preferably less than 7%, and even more preferably less than 2% of the preparation.

The purified compositions according to the invention preferably contain less than 5% by mass / mass (m / m), advantageously less than 3% m / m of impurities. It should be understood that references herein to "impurities" are to be understood to include unwanted reaction products that are not isomers formed during the synthesis and do not include residual solvents that remain from the process used in preparing the composition or excipients used in pharmaceutical preparations.

The term "essentially free" of a molecule means that the molecule is present in a composition only as an unavoidable impurity.

"Subject" means any animal, preferably a human patient, livestock, or domestic pet.

The "embryonic stem cell" (ES) is defined by its origin, that is, from one of the earliest stages of embryo development, called the blastocyst. Specifically, embryonic stem cells are derived from the inner cell mass of the blastocyst at one stage before it implants into the uterine wall. At this stage, the preimplantation embryo of the mouse is made up of 150 cells and consists of a sphere composed of an outer layer of cells (the trophectoderm), a fluid-filled cavity (the blastocellus), and a group of cells inside (the inner cell mass). Pluripotent ES cells can give rise to differentiated cell types that are derived from the three primary germ layers of the embryo (endoderm, mesoderm, and ectoderm).

The "adult" stem cell is an undifferentiated (unspecialized) cell found in differentiated (specialized) tissue; it can be renewed and specialized to produce all types of specialized cells from the tissue from which it originated. Adult stem cells are capable of self-renewal during the life of the body. Sources of adult stem cells have been found in the bone marrow, the bloodstream, the cornea and retina of the eye, the dental pulp of the tooth, the liver, the skin, the gastrointestinal tract, and the pancreas. Unlike embryonic stem cells, there are no identifiable adult stem cells that are capable of forming all the cells in the body. However, blood stem cells (derived from mesoderm) can generate a number of differentiated cells that include both skeletal muscle (also derived from mesoderm) and neurons (derived from ectoderm).

The term "substituted", as used herein, means any of the above groups (ie, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, homocycle, heterocycle, and / or heterocycloalkyl) wherein at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent ("= O"), two hydrogen atoms are replaced. When substituted, one or more of the above groups are "substituents". Substituents in the context of this invention include halogen, deuterium, tritium, boron, hydroxy, oxo, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle, and heterocycloalkyl , as well as a saccharide, --NRaRb, --NRaC (= O) Rb, -NRaC (= O) NRaNRb, --NRaC (= O) ORb, --NRaSÜ2Rb, --C (= O) Ra, C (= O) ORa, --C (= O) NRaRb, --OC ( = O) NRaRb, --ORa, --SRa, -SORa, --S (= O) ² Ra, --OS (= O) ² Ra and --S (= O) ² ORa. Furthermore, the above substituents may be further substituted with one or more of the above substituents, such that the substituent is substituted alkyl, substituted aryl, substituted arylalkyl, substituted heterocycle, or substituted heterocycloalkyl. Ra and Rb in this context can be the same or different and independently hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocycloalkyl, or substituted heterocycloalkyl. In the context of certain embodiments, a compound can be described as "unsubstituted," meaning that the compound does not contain additional substituents attached to the compound. An unsubstituted compound refers to the chemical composition of the compound without additional substituents, for example the compound does not contain protecting group (s). For example, unsubstituted proline is the amino acid proline although the amino group of proline can be considered disubstituted with alkyl groups.

Nicotinamide adenine dinucleotide pathway

Nicotinamide adenine dinucleotide (NAD or NAD +) is important as a coenzyme for different enzymes. Recent studies have shown that, when it is the cosubstrate of SIR2 (silent information regulator 2), NAD + has a role in the regulation of multiple biological processes, such as p53-regulated apoptosis, fat storage, resistance to stress and gene silencing. Without limiting the potential uses of the compositions described herein to a single theory, there are several pathways through which NAR is currently thought to be metabolized. Nicotinamide riboside (NAR) is known as a NAD + precursor for humans and yeast. It is capable of entering a natural pathway that leads to the biological synthesis of NAD + under the action of the enzyme nicotinamide riboside kinase (Nrk). NAR is converted to NMN by Nrk, which is then converted to NAD + by the enzyme nicotinamide mononucleotide adenylyltransferase (Nmnat) (see Figure 7). Alternatively, the nicotinamide riboside can enter the NAD metabolism through other metabolic pathways, which would include the action of enzymes that separate the remaining nicotinamide from the sugar. Such a pathway would include the action of phosphorylases that have been shown to degrade NAR in cells to form nicotinamide and ribose-1-phosphate. Nicotinamide is competent to enter the NAD + metabolism and is converted to NAD + by the action of the enzyme nicotinamide pyrophosphoribosyltransferase.

Sirtuins are class III histone deacetylases (HDAC) and are also ADP-ribosyl transferases. They deacetylate lysine residues in a new chemical reaction that consumes nicotinamide adenine dinucleotide (NAD +), releasing nicotinamide, O-acetyl-ADPribose (AADPR), and the deacetylated substrate. Altering intracellular levels of NAD + can improve the health of a cell, but the introduction of compounds that enter the metabolic pathways of NAD + can also be toxic to cells. In some embodiments, the invention relates to the use of the compounds described herein to manipulate NAD + levels, to modulate the activity of sirtuins and other ADP-ribosyl transferases, and to modulate IMPHD. These embodiments are used to destroy or weaken the defenses of cancer cells, or to promote the survival of neurons, myocytes, or stem cells through addition to growth media.

Nicotinic acid is an effective agent for controlling low-density lipoprotein cholesterol, increasing high-density lipoprotein cholesterol, and lowering triglyceride and lipoprotein (a) levels in humans. Although nicotinic acid treatment affects all key lipids in the desirable direction and has been shown to reduce mortality in target populations, its use is limited due to a side effect of heat and redness called flushing. Furthermore, nicotinamide protects against stroke injury in model systems, presumably due to multiple mechanisms, including increased mitochondrial levels of NAD +.

In some embodiments, the description refers to the use of the compounds described herein as agonists and antagonists of enzymes in the pathway of NAD + biosynthesis. In further embodiments, the NAR derivatives described herein are agonists, that is, they stimulate activities normally stimulated by naturally-occurring substances, of one or more Surtuins, preferably SIRT1 in humans or Sir2p in yeast. In additional embodiments, the NAR derivatives are antagonists of one or more sotuins.

Although the applicant does not intend the disclosure to be limited by any particular mechanism, it is believed to be useful because SIRT1 deacetylates transcription factors such as FOXOs, p53, and nuclear factor Kappa B (NFKB), which provide resistance to stress, apoptosis, and inflammatory responses. that improve the survival of the organism. SIRT1 sirtuin upregulates stress protection pathways by deacetylation of FOXO transcription factors, leading to increased transcription of GADD45 (DNA repair) and MnSOD (reactive oxygen detoxification). SIRT1 concomitantly down-regulates FOXO transcription of pro-apoptotic factors Fas and Bcl-2 mediators of cell death (BIM).

For example, the p53 network in non-activated normal situations is not functional, but is activated in cells in response to various signals, inhibiting abnormal cell growth and triggering programmed cell death. SIRT1 interacts with p53 and deacetylates the C-terminal regulatory domain. This activity downregulates the effects of transcriptional activation of p53 on target genes.

Chemical synthesis of nicotinoyl ribosides

Due to its biological importance, a number of chemical approaches have been used to prepare nicotinamide riboside (NAR). See Franchetti et al., Bioorganic & Medicinal Chemistry Letters 14, 4655-4568 (2004) and Tanimori et al., Bioorganic & Medicinal Chemistry Letters 12, 1135-1137 (2002). However, there is still no systematic way to synthesize NAR analogs. One embodiment of the invention relates to an efficient method for stereoselectively synthesizing p-NAR and its various derivatives in a container with high throughput. Compared to previous methods, this stereoselective method is more efficient and offers more possibilities to form various NAD + analogs with a similar procedure.

The synthesis of nicotinoyl riboside compounds was initiated with the preparation of a preferred intermediate, ethyl 1 - [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate. From this, O-alkyl derivatives of nicotinic acid riboside were prepared. Refluxing the protected sugar, 1,2,3,5-tetra-O-acetyl-pD-ribofuranose with ethyl nicotinate (1.2 equiv.) In methylene chloride under the catalysis of TMSOTf (1 equiv) stereoselectively formed the isomer for good yield (> 90%). One of the advantages of this method is that there is no need to go through the silylation of nicotinamide. After evaporation of the solvent, ethyl 1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate can be used directly for the next step. P-NAR and its derivatives are extremely soluble in water. Extraction of ethyl acetate from the water layer was able to remove most of the non-polar by-products and give 95% pure products according to TLC. Ethyl 1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate was also treated with NaOCH3 / MeOH at -20 ° C to form O-methyl nicotinate riboside . Other O-alkyl nicotinate ribosides were prepared in a similar manner. O-alkyl nicotinate ribosides can be hydrolyzed with esterase (from pig liver, Sigma) at pH = 7.0 to provide clean nicotinic acid riboside. Reverse HPLC was used to detect and purify the products.

The thermodynamic study showed the reaction of ethyl 1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate and NH3 / MeOH at -20 ° C gave the product 1- [ Methyl 3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate, instead of p-NAR. However, without exhausting, the reaction could go further to finally form p-NAR after it was restored to 4 ° C. This could be explained thermodynamically. Since the amide is thermodynamically more stable than the ester, at a higher temperature, the more stable p-NAR would be the primary product. However, reaction with NaOCH3 at a lower temperature, -20 ° C, kinetically favors the formation of O-methyl nicotinate riboside.

The preparation of N-alkyl p-NAR derivatives starts from 2,3,5-tri-O-acetyl-P-phenyl nicotinate riboside (see Figure 5) and an activated phenyl ester. Incubation of nicotinic chloride with phenol in THF overnight at room temperature produced quantitatively pure phenyl nicotinate, which was then treated with 1,2,3,5-tetra-O-acetyl-pD-ribofuranose in methylene chloride under the catalysis of TMSOTf to 2,3,5-tri-O-acetyl-p-phenyl nicotinate riboside. The riboside 2,3,5-Tri-O-acetyl-p-phenyl nicotinate could be further transformed to prepare multiple derivatives of N-alkyl NR with the addition of various primary or secondary amines. It was also considered that the intermediate ethyl ester was used to construct the derivatives of N-alkyl-NAR, since in the literature the ester-amide exchange has been considered a versatile and simple way to prepare amides. However, the inventors' thermodynamic study (see Figure 6) showed that incubation of the intermediate ethyl ester with N-methylamine in methanol at -20 ° C formed the O-methyl nicotinate riboside as a product, instead of the N- expected methyl nicotinamide riboside. The MALDI-MS of the product showed the molecular weight at 270, which is a mass more than the expected 269. The m / z peak was also detected at 138 instead of 137, which was due to the methyl nicotinate piece after that the C1'-N1 bond of the methyl ester was broken. All information suggests that a transesterification rather than an ester-amide exchange occurs. At low temperature, both the transesterification and the ester-amide reactions were very slow, so that the selectivity of the reaction was kinetically controlled. Transesterification was more kinetically favored than ester-amide exchange due to the higher concentration of the solvent, methanol. Since the amide is thermodynamically more stable than the ester, we assume that the selectivity of the reaction at the higher temperature would be thermodynamically controlled to form the more stable amide product. Therefore, the same reaction mixture was incubated at 4 ° C. However, after one night, the riboside was totally decomposed, and N-methylamine and free sugar were formed, which did not happen in the preparation of p-NAR with the use of NH3 / MeOH at all. The decomposition may be due to the fact that N-alkylamines are more basic / neutrophilic than free ammonia, which tends to attack the C-1 'position of the riboside and cause the C1-N1 bond to break.

To avoid decomposition, another reaction solvent, trifluoroethanol (TFE), has been tried. TFE has a pKa of 12.5, which makes it more acidic compared to solvents like methanol or ethanol. This acidity of TFE decreases the neutrophilia of the N-alkylamines and thus helps to stabilize the C1'-N1 bond. The ethyl ester reaction was then carried out in trifluoroethanol at 4 ° C for days. The acidity of trifluoroethanol was found to also moderate the nucleophilicity / basicity of N-methylamine such that the ester-amide exchange reaction takes weeks to complete. The ester-amide exchange of the phenol ester in trifluoroethanol at 4 ° C could be completed overnight. Interestingly enough, it was also found that the 2'-, 3'- and 5'-O-acetyl groups in sugar exhibit different rates of deacetylation under the same basic conditions. After treatment with N-methylamine at 4 ° C overnight, MALDI-MS has shown the molecular weight of the isolated product as 311 instead of the expected 269. The 1H-NMR also agreed that the 5'-O-acetyl group does not it had been removed from the molecule. This method of selective deprotection in fact presents a convenient opportunity for a selective modification at the 5 'position of ribose. After 4N NH3 / MeOH was added to the reaction mixture, the reaction was completed after an overnight incubation at -20 ° C, and gave the expected deprotected product. All other N-alkyl nicotinamide ribosides have also been prepared by a similar procedure.

Compositions for administration

Compositions comprising the active compounds (nicotinamide riboside derivatives) include nutritional / dietary supplements and bulk drug compositions useful in the manufacture of pharmaceutical compositions (eg, impure or non-sterile compositions) and pharmaceutical compositions (ie, compositions that are suitable for administration to a subject) that can be used in the preparation of unit dosage forms. Such compositions optionally comprise a prophylactic or therapeutically effective amount of a prophylactic and / or therapeutic agent described herein or a combination of those agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the disclosure comprise a prophylactically or therapeutically effective amount of the active compound and another therapeutic or prophylactic agent, and a pharmaceutically acceptable carrier. These compositions can contain between 0.1-99% of the active ingredient.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the US Pharmacopeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical carriers can be saline, acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. When administered to a subject, pharmaceutically acceptable carriers are preferably sterile. Water can be the vehicle when the active compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, and the like. The present compositions, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The present compositions may take the form of solutions, suspensions, emulsions, tablets, pills, granules, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions or any other form suitable for their use. use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see, for example, US Patent No. 5,698,155).

In a preferred embodiment, the active compound and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to humans. Typically, the active compound for intravenous administration are solutions in sterile isotonic aqueous buffer. When necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dosage form, for example, as a freeze-dried dry powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. . When the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the active compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

Compositions for oral administration may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs, for example. Orally administered compositions may contain one or more optional agents, eg, sweetening agents such as fructose, aspartame, or saccharin; flavoring agents such as peppermint, wintergreen or cherry oil; coloring agents; and preservatives, to provide a pharmaceutically acceptable preparation. In addition, when in tablet or pill form, the compositions can be coated to delay disintegration and absorption from the gastrointestinal tract, thus providing sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active actuation compound are also suitable for oral administration of the active compound. In these rear platforms, the actuation compound absorbs fluid from the environment surrounding the capsule, which swells to displace the agent or agent composition through an opening. These delivery platforms can provide an essentially zero order delivery profile as opposed to the enriched profiles of release formulations. immediate. A time delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such carriers are preferably of pharmaceutical grade.

Furthermore, the effect of the active compound can be delayed or prolonged by a suitable formulation. For example, a slowly soluble tablet of the active compound can be prepared and incorporated into a tablet or capsule. The technique can be improved by making granules of different dissolution rates and filling the capsules with a mixture of the granules. Tablets or capsules can be coated with a film that will resist dissolution for a predictable period of time. Even parenteral preparations can be made long-acting by dissolving or suspending the compound in oily or emulsified vehicles that allow it to only slowly disperse in the serum.

Compositions for use in accordance with the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.

Therefore, the compound and optionally another therapeutic or prophylactic agent and its physiologically acceptable salts and solvates can be formulated in pharmaceutical compositions for administration by inhalation or insufflation (through the mouth or nose) or orally, parenterally or mucosa ( such as buccal, vaginal, rectal, sublingual administration). In one embodiment, local or systemic parenteral administration is used.

For oral administration, the compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinized cornstarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc, or silica); disintegrants (eg, potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavorings, colors and sweeteners, as appropriate.

Preparations for oral administration can be suitably formulated to provide controlled release of the active compound.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compositions for use according to the present disclosure are conveniently administered in the form of an aerosol presentation from pressurized containers or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin can be formulated for use in an inhaler or insufflator containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

The compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile pyrogen-free water, prior to use.

The compositions can also be formulated into rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations previously described, the compositions can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, pharmaceutical compositions can be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, eg, as a sparingly soluble salt.

In other embodiments of the disclosure, radiation therapy agents such as radioactive isotopes may be administered orally as liquid in capsules or as a beverage. Radioactive isotopes can also be formulated for intravenous injection. The skilled oncologist can determine the preferred formulation and route of administration.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The package may comprise, for example, a metal or plastic foil, such as a blister package. The package or dispensing device may be accompanied by instructions for administration.

In certain preferred embodiments, the pack or dispenser contains one or more unit dosage forms containing no more than the recommended dosage formulation as determined in Physician's Desk Reference (56th edition, 2002).

Methods for administering the active compound and optionally another therapeutic or prophylactic agent include, but are not limited to, parenteral (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (eg, routes) administration. intranasal, rectal, vaginal, sublingual, buccal or oral). In a specific embodiment, the active compound and optionally other prophylactic or therapeutic agents are administered intramuscularly, intravenously, or subcutaneously. The active compound and optionally another prophylactic or therapeutic agent can also be administered by infusion or bolus injection and can be administered in conjunction with other biologically active agents. Administration can be local or systemic. The active compound and optionally the prophylactic or therapeutic agent and their physiologically acceptable salts and solvates can also be administered by inhalation or insufflation (through the mouth or nose). In a preferred embodiment, local or systemic parenteral administration is used.

In specific embodiments, it may be desirable to administer the active compound locally to the area in need of treatment. This can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, for example, in conjunction with a wound dressing after surgery, by injection, by catheter, by suppository or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including membranes, such as elastic membranes or fibers. In one embodiment, administration may be by direct injection into the site (or anterior site) of an atherosclerotic plaque tissue.

Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or by perfusion in a fluorocarbon or synthetic lung surfactant. In certain embodiments, the active compound can be formulated as a suppository with traditional binders and carriers, such as triglycerides.

In another embodiment, the active compound can be administered into a vesicle, in particular a liposome.

In yet another embodiment, the active compound can be administered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, polymeric materials can be used.

The amount of the active compound that is effective in treating or preventing heart conditions can be determined by standard investigative techniques. For example, the dosage of the active compound that will be effective in treating or preventing heart conditions can be determined by administering the active compound to an animal in a model such as, for example, animal models known to those of skill in the art. Additionally, in vitro assays can optionally be employed to help identify optimal dosage ranges.

The selection of a particular effective dose can be determined (eg, through clinical trials) by one of skill in the art based on consideration of several factors that will be known to those of skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the body mass of the subject, the immune status of the subject, and other factors known to those of skill in the art.

The precise dose to be employed in the formulation will also depend on the route of administration and the severity of disease-related wasting, and should be decided in accordance with the judgment of the physician and the circumstances of each subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The dose of the active compound to be administered to a subject, such as a human, is quite variable and can be subject to independent judgment. It is often practical to administer the daily dose of the active compound at various times of the day. However, in any given case, the amount of the active compound administered will depend on factors such as the solubility of the active component, the formulation used, the condition of the subject (such as weight) and / or the route of administration.

The general range of effective amounts of the active compound alone or in combination with other prophylactic or therapeutic agent (s) is from about 0.001 mg / day to about 1000 mg / day, plus preferably from about 0.001 mg / day to 750 mg / day, more preferably from about 0.001 mg / day to 500 mg / day, more preferably from about 0.001 mg / day to 250 mg / day, more preferably from about 0.001 mg / day to 100mg / day, more preferably from about 0.001mg / day to 75mg / day, more preferably from about 0.001mg / day to 50mg / day, more preferably from about 0.001mg / day to 25mg / day, more preferably from about 0.001 mg / day to 10 mg / day, more preferably about 0.001 mg / day to 1 mg / day. Of course, it is often practical to administer the daily dose of compound in portions, at various times of the day. However, in any given case, the amount of compound administered will depend on factors such as the solubility of the active component, the formulation used, the condition of the subject (such as weight) and / or the route of administration.

A popular cancer drug is taxol. Typical dosage ranges for taxol include less than 10 mg to 100 mg or more. Particular doses of taxol include about 5mg, 10mg, 20mg, 40mg, 50mg, 60mg, 80mg, 100mg, 150mg, 200mg. These are generally daily doses. In general, higher doses are less preferred due to possible gastric disturbances. Therapeutic doses can range from 40 to 80 mg per day when tolerable by a subject.

The disclosure provides any method of administering lower doses of known agents (eg, taxol) than previously thought to be useful for the prevention or treatment of cancer. EXAMPLES

Example 1: Synthesis of 2 ', 3', 5'-triacetyl ethyl nicotinate riboside (1- [3,4-diacetyloxy-5- (acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate)

One equivalent of trimethylsilyl trifluoromethanesulfonate (TMSOTf) in ethyl nicotinate (0.9 ml, 6.6 ml) and 1,2,3,5-tetra-O-acetyl-pD-ribofuranose (1.4 g , 4.4 mmol) in 50 mL of anhydrous methylene chloride at room temperature. The mixture was then heated under reflux for 8 hours. TLC stained with H ² SO ⁴ in MeOH showed the disappearance of 1,2,3,5-tetra-O-acetyl-pD-ribofuranose, and an almost pure product. The product could be used directly for the next step after the methylene chloride was evaporated.

Example 2: Synthesis of p-NAR (Nicotinamide Riboside)

2 ', 3', 5'-triacetyl ethyl nicotinate riboside (180 mg, 0.44 mmol) in 4.7 ml of 4N NH ³ / MeOH on ice was added. After mixing well, the reaction was stored at 4 ° C overnight. After removing methanol in vacuo, the residue was dissolved in water and extracted with ethyl acetate three times to remove the non-polar impurity. The water layer was then concentrated and injected into reverse HPLC for purification (Waters xTerra Prep RP18® column, 2 ml / min, 20 mM ammonium acetate).

Example 3: O-alkyl p-nicotinate ribosides

2 ', 3', 5'-triacetyl ethyl nicotinate riboside (25 mg, 0.61 pmol) in 0.9 ml NaOMe / MeOH (255 mM) or NaOEt / EtOH (312 mM) was added on ice. After mixing well, the reaction was stored at -20 ° C overnight. The reaction was quenched with the addition of acetic acid to pH = 7. After removing the organic solvent in vacuo, the residue was dissolved in water and extracted with cyclohexane to remove non-polar impurities. The water layer was then concentrated and injected into reverse HPLC for purification.

Reference example 4: N-alkyl p-nicotinamide ribosides

Nicotine chloride (1.78 g, 10 mmL) was added to phenol (1.29 g, 12 mmL) in 10 mL of THF. Triethylamine (5 ml) and pyridine (5 ml) were then added to the mixture, which was then stirred at room temperature overnight. After the THF was evaporated, the mixture was then washed with ethyl acetate in water three times. The organic layer was then purified with a silica column (Hexane: EtOAc = 4: 1) to give the pure product. The riboside was prepared according to the procedure provided in Example 1. This phenol ester (25 mg, 0.61 pmol) was added to various amines (400 pM) in 0.9 ml of trifluoroethanol. After mixing well, the reaction was stored at 4 ° C overnight. The next day, 4N NH ³ / MeOH was added to the reaction mixture, and the reaction was stored at -20 ° C overnight. After the reaction was quenched with the addition of HCl to make the pH <7, the organic solvent was removed in vacuo. The residue was dissolved in water and isolated with a disposable Octadecyl-C18 extraction.

Table 2

Inlet (Figure 1) Intermediate Temp. Product Yield (%)

Solvent

Tcmp

3 R '= C ² H ⁵ MeOH 4 ° CR = NH ² 85

4 R '= C ² H ⁵ MeOH -20 ° CR = OCH ³ 85

5 R '= C ² H ⁵ EtOH -20 ° CR = OC ² H ⁵ 81

6 R '= C ² H ⁵ R = OH 80

9 R '= C6H5 TFE 4 ° CR = NHCH ³ 80

10 R '= C6H5 TFE 4 ° C R = NHC2H5 80

11 R '= C6H5 TFE 4 ° C R = NHCH2CH = CH2 80

12 R '= C6H5 TFE 4 ° C R = NHC2H4NH2 55

13 R '= C ⁶ H ⁵ TFE 4 ° CR = NH (CH3) 2 52

Reference example 5

Synthesis of p-nicotinic acid riboside (1- [3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] pyridine-5-carboxylic acid) 1- [3,4-diacetyloxy-5- ( methyl acetyloxymethyl) oxolan-2-yl] -pyridine-3-carboxylate in phosphate buffer (150 mM, pH = 7.0). 10 µl of esterase was added to the mixture and mixed well. The reaction was carried out at 25 ° C overnight. HPLC injection showed a 98% pure product.

Reference example 6. Increase in cellular levels of NAD + produced with medium enriched with Nicotinamide Riboside (NAR)

Mouse embryonic stem cells were seeded into cell culture flasks (250 ml, 75 cm2) and grown to 50% confluence within 48 hours after passage. At 48 hours 2 flasks were set apart as controls and 2 flasks were used to determine the effect of NAR on NAD + biosynthesis. The cells were treated with 41 M HClO and a specific amount of 18O-NAM (nicotinamide mononucleotide) and 18O-NAD +. The samples were fractionated by HPLC and the fractions containing NAM and NAD + were analyzed by ESI-MS and MALDI-MS, respectively. The maximum ratio (16O / 18O) was used to quantify amounts of NAM and NAD + in each sample. Cells treated with NAR were found to show a higher concentration of NAD + compared to controls. According to our measurement, the NAD + level in NAR-treated cells is 3886.6 pmoL / mg of cellular protein and nucleic acid content, and the NAD + in control cells is only 1782.2 pmoL / mg of cellular protein and nucleic acid content. Cellular protein and nucleic acid content were measured separately (see Figure 3).

Reference example 7. Effect of nicotinamide riboside (NAR) on mms-mediated cell death Mouse embryonic stem cells are transferred to 7 flasks (250 ml, 75 cm2) with 20 ml of medium [For 500 ml: Dulbecco's modification of Eagle's medium, 2 mM glutamine, 10% ES 10% graded fetal calf serum %, penicillin / streptomycin (5 ml 100 x), non-essential amino acids (5 ml 100 x), sodium pyruvate (5 ml 100 x), 4 μl of beta-mercapto-ethanol and final concentration of the leukemia inhibitory factor 1000 units / mL], named from no. 1 to no. 7. After 2 days of incubation at 37 ° C with 5% CO ² , NAR was added to flask # 3, 5, 7 to make the final concentration to 1 mM, and the cells were incubated for another day. Mms (200 mM) was added to flask # 2, 3, 4, 5, 6, 7 to obtain a final concentration of 1 mM. Cell flask no. 1 is kept as a control and is used for the step. After 3 hours of incubation, the fresh medium was changed to flask no. 4, 5, 6, 7. And Cells no. 2 and 3 are harvested. After 6 hours, the mms treatment, Cell no. 4, 5. And the Cells do not. 6, 7 are harvested at 9 h of mms treatment. Harvested cells (live cells) are counted to determine the percentage of kill with trypan blue (see figure 2).

Reference example 8. Cell survival

Mouse embryonic stem cells are transferred to 2 flasks (250 ml, 75 cm2) with 20 ml of medium, designated no. A and no. B. After 2 days of incubation at 37 ° C with 5% CO ² , NAR was added to flask no. A to obtain the final concentration at 1 mM, and both cells were incubated for another day. Mms (200 mM) was added to both flasks #. A and no. B to obtain a final concentration of 1 mM. After 3 hours of incubation, each cell culture (with / without NR treatment) is transferred to two new flasks with / without 1 mM NAR. After two days of incubation, count the culture survival in each of these 4 new flasks: mms / control medium, mms / NAR medium; medium mms NAR / control; half mms NAR / NAR. It was found after mms treatment that the cultured cells were seriously damaged and had only very poor survival after passage. However, compared to others, the NAR-treated cell culture provides the highest culture numbers. Cell count by hemocytometry was used to determine survival versus cell number (see Figure 4).

Reference Example 9. Effects of Statins on Cell Viability and Mitigation of Nicotinamide Riboside Toxicity

Equal amounts of mouse embryonic stem cells are passed into two 6-well tissue culture plates (10 cm2 each well) with 3 ml of medium [For 500 ml: Dulbecco's modification of Eagle's medium, 2 mM glutamine, Fetal Bovine Serum ES 10% graded to 10%, penicillin / streptomycin (5 ml 100 x), non-essential amino acids (5 ml 100 x), sodium pyruvate (5 ml 100 x), 4pL beta-mercapto-ethanol and final concentration of inhibitory factor of leukemia 1000 units / ml], named from n. 1 to No. 6. After 2 days of incubation at 37 ° C with 5% CO ² , different concentrations of lovastatin 0, 50, 250, 800, 2000 and 10,000 nM were added to wells 1-6 in each plate, respectively. The two plates were named NR and not NR to represent treatment and lack of treatment with the compound nicotinamide riboside (NR). Each well on the NR plate was treated with 10 pL of 150 mM NR to make a final concentration of NR in each well of 500 pM. At 2, 4 and 7 days the cells were analyzed by cell counting and MTT assay after trypsinizing the cells to detach them from the plates. At 2 and 4 days the cells were reseeded from each individual well into new wells in fresh 6-well plates and were immediately treated with the same concentration of NR and statins as the cells in the source well. The seeded cells were identical in number of the viable cells in each case as determined by counting the cells with an appropriate dilution of the trypsinized cells from each well.

Cell viability at each time point was plotted against statin concentration to determine the IC50 of the statin in the plate with NR and without NR where IC50 is defined as the statin concentration that reduces cell viability by 50% in the experimental conditions. Statin typically reduces cell viability by 50% at a concentration between 130nM-300nM at each time point for cells not treated with NR (Figure 8). In the presence of NR, the IC50 of the statin increases moderately after 2 days of treatment and two to three times after 4 and 7 days of treatment (Figure 8). This shows that NR mitigates statin toxicity in a significant way with NR available in cellular media.

Cell viability measured by cell count and MTT assay is significantly higher in the group treated with NR versus no NR at virtually every statin concentration, resulting in a higher IC50 for statins in the presence of NR (566 nM, determined by cell count; 656 nM determined by MTT assay) vs. absence of NR (248 nM, determined by cell count; 216 nM determined by MTT assay). Notably, at the highest measured statin concentration, 10 pM, significant percentages of viable cells remain in the NR-treated group, as measured by both cell count and MTT assay, while cell viability in the no-NR group is nearly completely unfeasible. The data analysis used was analogous in the results of days 2 and 4 that are shown graphically in FIG. 8.

Reference example 10. Evaluation of different nicotinate riboside derivatives as bioavailable forms of niacin that stimulate the synthesis of NAD + in cells

Hela cells were seeded in a 6-well plate at 37 ° C with 5% CO ² for 24 hours. The synthesized compounds (NAR: nicotinic acid riboside, O-ethyl-NR: ethyl nicotinate riboside, Intermediate: TAENR, triacetyl ethyl nicotinate riboside, see Figure 10) were added to cells separately after 24 hours (no compound was added for control). The cells were then incubated for another 24 hours before harvesting. Cells were trypsinized and an aliquot was removed for counting. Cells were pelleted and then dissolved in 100 ul 7% perchloric acid, and 535.5 pmoL 18O-NAD + (95% isotopically enriched) was added to each sample. After mixing well, the samples were sedimented and then neutralized with NaOH to make pH = 7 ~ 8. The samples were then centrifuged for 3 minutes and the supernatant (100 ul) was injected onto a C-18 column (L2450 diode array detector from Hitachi HPLC pump L2130) and separated to isolate the peak containing NAD +. The elution was collected during the NAD + peak and the solution was frozen and lyophilized. The sample was analyzed by MALDI-MS in positive ion detection mode to determine the content of NAD + (Table 3) by measuring the peaks m / z = 664 (16O-NAD + and 666 (18O-NAD +).

The experiment suggests that different forms of nicotinate ribosides are available as metabolizable sources of niacin and that these newly composed chemical forms of niacin have the effect of substantially increasing NAD + concentrations in cells as determined by a quantitative MS assay. Increases in NAD + concentrations within cells can lead to clinical benefits. The minimum increase in the concentration of NAD + in the cells that we observed was 618 pmol per million cells (Table 3, and bar graph Figure 9) in untreated cells up to 1004 pmol per million cells in the case of treatment with 500 micromolar of the O-ethyl nicotinate riboside compound This represents a 62% increase over the control of the NAD + content. Increasing the concentration of this compound to 800 micromolar led to an increase in the concentration of NAD + of approximately three times (Table 3). Similarly, nicotinic acid riboside and fully esterified tri-acetylethylnicotinate riboside compound were able to increase NAD + concentrations in cells by 60% and three-fold respectively (Table 3). These data demonstrate that these derivatives are metabolized by the cells and increase the NAD + concentrations in the cells substantially (60-300%). These compounds increase intracellular NAD +.

Table 3: NAD + content (pmoL) in ES cells after being treated with different compounds

NAR: nicotinic acid riboside, O-ethyl-NR: ethyl nicotinate riboside, TAENR, riboside triacetyl-ethyl-nicotinate

Reference Example 11. Reduction of Statin Toxicity

ES cells were grown in four 6-well plates at 37 ° C with 5% CO ² for 24 hours. The cells were then treated with 0, 800 or 5000 nM lovastatin (final concentration) and were also treated with one of 500 gM NAR, 1 mM OENR, 1 mM TAENR or no compound. After 48 hours, cell media was removed and cells were harvested by trypsinization. Cells were counted by hemocytometry and using propidium iodide staining to assess viability. Cells were counted for no statin treatment and 800 nM and 5 gM statin treatment in each group. The percentage of cell death was determined by the viable cells in each group divided by the cell numbers in the corresponding group not treated with statins. The cell counts were similar in the controls of each group (without statins) indicating a low toxicity of the nicotinate riboside compounds added to the treated cells.

Figure 10 shows data suggesting that statin toxicity may be mitigated by nicotinate riboside compounds. The lower percentages of cell death show that nicotinate riboside compounds prevent cells from succumbing to toxic statin treatment. These compounds are believed to be able to enter NAD + metabolism as demonstrated by our study of the potency of these molecules as precursors to NAD + and thereby protect cells from stress or toxicity. Therefore, various degenerative disorders that result from disease processes can be treated using these compounds.

Reference Example 12. Toxicity of Selected Nicotinamide Riboside Derivatives

To 96-well plates, mouse ES cells were incubated in cell culture medium for a period of 24 hours without compound (control) dimethyl nicotinamide riboside (1 mM) or N-allyl nicotinamide riboside (1 mM). After a 24 hour period, cells were treated with MTT reagent and further incubated at 37 degrees. centigrade for an additional three hours. After this time, the media was removed by pipetting and DMSO was added to solubilize the dye. The purple color was measured at 590 nm in a plate reader to assess cell viability. All samples were run in duplicate and the mean MTT reading was compared to the mean reading versus the control. N-allyl-nicotinamide-riboside is able to reduce cell proliferation compared to controls that are not treated with the compound (Figure 12).

Example 13. Synthesis and chemical characterization

All organic solvents and reagents were purchased from Sigma-Aldrich Corporation and VWR Scientific and used without further purification. The HPLC system consists of a Hitachi EZChrom Elite HPLC system with an L2450 diode array as a detector. The purification system employed a mobile phase system involving 20 mM ammonium acetate buffer at a flow rate of 2.0 ml / min and a Waters Delta PAK C18 column (15 pm, 300 x 7.8 mm). MALDI mass spectra were obtained from the Rockefeller University Proteomics Resource Center using the DERP MALDI-TOF PerSeptive Voyager Mass Spectrometer. HRMS spectra were obtained from the Hunter College Mass Spectrometry Facility. The 1H and 13C NMR spectra were recorded on the Bruker NMR spectrometer operating at 400 MHz (DPX Advance), respectively. The 1H and 13C chemical shifts are expressed in ppm relative to standard deuterium solvent peaks.

Phenyl nicotinate:

Nicotinic chloride (1.78 g, 10 mmoL) was added to phenol (1.29 g, 12 mmL) in 10 ml of tetrahydrofuran (THF). Triethylamine (5 ml) and pyridine (5 ml) were then added to the mixture, which was then stirred at room temperature overnight. After evaporating the THF, the mixture was washed with ethyl acetate in water three times. The ethyl acetate (EtOAC) layer was then purified with a silica column (Hexane: EtOAc = 4: 1) to give the pure product. 1H NMR (400 MHz, CDCta): 5 = 9.40 (s, 1H), 8.86 (d, 1H), 8.46 (d, 1H), 7.45 (m, 3H) , 7.32 (d, 1H), 7.24 (m, 2H).

2 ', 3', 5'-Triacetyl ethyl nicotinate riboside (2):

1 equiv. Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was added slowly in ethyl nicotinate (0.9 ml, 6.6 ml) and 1,2,3,5-tetra-O-acetyl-pD-ribofuranose (1.4 g, 4 , 4 mmol) in 50 ml of anhydrous methylene chloride at room temperature. The mixture was then heated under reflux for 8 hours. TLC (MeOH: MeOH: TEA = 5: 0.3: 0.05) stained with 10% H ² SO ⁴ in MeOH showed the disappearance of ribofuranose, and an almost pure product. Product 2 was then used directly for the next step after the methylene chloride was evaporated. MS: M / Z (%): 410.06 (14, [M] +), 259.0 (100), 138.9 (22).

2 ', 3', 5'-Triacetyl phenyl nicotinate riboside (8):

1 equiv. Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was slowly added to phenyl nicotinate (100 mg, 0.41 mmol) and 1,2,3,5-tetra-O-acetyl-pD-ribofuranose (159 mg, 0.5 mmol) in 15 ml of anhydrous methylene chloride at room temperature. The mixture was then heated under reflux for 5 hours. TLC (MeOH: MeOH: TEA = 5: 0.3: 0.05) stained with 10% H ² SO ⁴ in MeOH showed the disappearance of 1, and an almost pure product. The product could be used directly for the next step after the methylene chloride was evaporated. MS: M / Z (%): 458.1 (12, [M] +), 139.0 (64), 200.0 (76).

p-nicotinamide riboside (3)

Compound 2 (180 mg, 0.44 mmol) was added to 4.7 ml of 4N NH3 / MeOH on ice. After mixing well, the reaction was stored at 4 ° C overnight. After removing the methanol in vacuo, the residue was dissolved in water and extracted with ethyl acetate three times to remove non-polar impurities. The water layer was then concentrated and injected onto reverse HPLC for purification. 1H ⁿ M ^r (400 MHz, D ² O): 5 = 9.60 (s, 1H), 9.31 (d, 1H), 8.92 (d, 1H), 8.16 (t , 1H), 6.06 (d, 1H), 4.31 (m, 2H), 4.19 (t, 1H), 3.90 (dd, 1H), 3.74 (dd, 1H) . 13C NMR (300 MHz, D ² O): 5 = 146.0, 143.0, 141.3, 128.7, 100.2, 88.1, 77.7, 70.2, 60.6. MS: 255 (67, [M] +), 133.0 (4), 123.0 (82). HRMS (ESI) m / z [M] + calcd for C ¹¹ H ¹⁵ N ² O ⁵ 255.09755, 255.09801 found.

O-alkyl p-nicotinic riboside (4, 5):

Compound 2 (25 mg, 0.61 gmol) was added to 0.9 ml of NaOMe / MeOH (255 mM, to form 4) or NaOEt / EtOH (312 mM, to form 5) on ice. After mixing well, the reaction was stored at -20 ° C overnight. The reaction was quenched with the addition of acetic acid to pH = 7. After removing the organic solvent in vacuo, the residue was dissolved in water and extracted with cyclohexane to remove non-polar impurities. The water layer was then concentrated and injected into reverse HPLC for purification.

O-methyl p-nicotinic riboside (4): 1 H NMR (400 MHz, D ² O): 5 = 9.74 (s, 1 H), 9.24 (d, 1 H), 9.05 (d , 1H), 8.23 (t, 1H), 6.21 (d, 1H), 4.42 (m, 2H), 4.29 (t, 1H), 3.99 (m, 4H ), 3.84 (dd, 1H), 13C NMR (300 MHz, D ² O): 5 = 147.8, 144.0, 142.2, 129.0, 100.5, 87.9, 77, 8, 69.9, 60.4, 54.4. MS: m / z (%): 270.1 (42, [M] +), 152.1 (81), 138.0 (92). HRMS (ESI) m / z [M] + calculated for C ¹² H ¹⁶ NO6270.09721, found 270.09737.

O-ethyl p-nicotinic riboside (5): 1H NMR (400 MHz, D ² O): 5 = 9.86 (s, 1H), 9.37 (d, 1H), 9.20 (d, 1H), 8.37 (t, 1H), 6.35 (d, 1H), 4.55 (m, 4H), 4.44 (t, 1H), 4.15 (dd, 1H), 3.98 (dd, 1H), 1.49 (t, 3H). 13C NMR (300 MHz, D ² O): 5 = 148.9, 145.0, 143.5, 129.8, 101.7, 88.8, 79.0, 70.8, 65.7, 61 , 4, 15.0. MS: m / z (%): 284.1 (77, [M] +), 256.0 (6), 212.0 (83), 133.0 (7), 124.0 (14). HRMS (ESI) m / z [M] + calculated for C ¹³ H ¹⁸ NO ⁶ 284.11286, found 284.11339.

P-nicotinic acid riboside (6)

Compound 5 was dissolved in phosphate buffer (150 mM, pH = 7.0). 10 µl of esterase was added to the mixture and mixed well. The reaction was carried out at 25 ° C overnight. HPLC injection showed that the purity of the product is above 98%. 1H NMR (400 MHz, D ² O): 5 =; MS: m / z (%): 256.1 (3, [M] +), 228.0 (80), 207.1 (42), 146.1 (17), 124.0 (23).

Derivatives of N-alkyl p-NAR (9, 10, 11, 12, 13):

Compound 8 (25 mg, 0.61 mol p) was added at various amine (400 p M) in 0.9 ml of trifluoroethanol. After mixing well, the reaction was stored at 4 ° C overnight. The next day, 4N NH ³ / MeOH was added to the reaction mixture, and the reaction was stored at -20 ° C overnight. After the reaction was quenched with the addition of HCl to make the pH <7, the organic solvent was removed in vacuo. The residue was dissolved in water and isolated with a disposable Octadecyl-C18 extraction column.

N-methyl p -nicotinamide riboside (9): 1H NMR (400 MHz, D ² O): 5 = 9.58 (s, 1H), 9.28 (d, 1H), 8.95 (d, 1H), 8.30 (t, 1H), 6.27 (d, 1H), 4.75 (1H), 4.53 (m, 1H), 4.39 (t, 1H ), 4.08 (dd, 1H), 3.93 (dd, 1H), 3.00 (s, 3H). 13C NMR (300 MHz, D ² O): 5 = 145.3, 142.4, 140.0, 128.4, 100.0, 87.8, 77.5, 69.9, 60.2, 27 ,5. MS: m / z (%): 269.1 (13, [M] +), 248.0, 212.0, 137.0 (100). HRMS (ESI) m / z [M] + calculated for C ¹² H ¹⁷ N ² O ⁵ 269.11320, found 269.11378.

N-ethyl p -nicotinamide riboside (10): 1H NMR (400 MHz, D ² O): 5 = 9.57 (s, 1H), 9.27 (d, 1H), 8.95 (d, 1H), 8.29 (t, 1H), 6.27 (d, 1H), 4.52 (m, 2H), 4.38 (t, 1H), 4.07 (dd, 1H), 3.92 (dd, 1H), 3.49 (q, 2H), 1.26 (t, 3H). 13C NMR (300 MHz, D ² O): 5 = 145.8, 142.7, 140.3, 129.0, 100.5, 88.0, 77.8, 70.0, 60.6, 36 , 0, 13.4. MS: m / z (%): 283.1 (93, [M] +), 227 (50), 207.1 (100), 151.1 (44).

N-allyl p -nicotinamide riboside (11): 1H NMR (400 MHz, D ² O): 5 = 9.59 (s, 1H), 9.28 (d, 1H), 8.98 (d, 1 H), 8.30 (t, 1H), 6.27 (d, 1H), 5.99 (m, 1H), 5.27 (m, 1H), 4.52 (m, 2 H), 4.38 (t, 1H), 4.08 (m, 3H), 3.92 (dd, 1H). 13C NMR (300 MHz, D ² O): 5 = 146.2, 143.5, 140.7, 133.7, 129.4, 117.3, 100.9, 88.4, 78.2, 70 , 8, 61.0, 43.5. MS: m / z (%): 295.1 (63, [M] +), 163.1 (98), 133.1 (5), 123.1 (88).

N-ethanol p -nicotinamide riboside (12): 1H NMR (400 MHz, D ² O): 5 = 9.55 (s, 1H), 9.24 (d, 1H), 8.93 (d, 1H) , 8.24 (t, 1H), 6.22 (d, 1H), 5.99 (m, 1H), 4.48 (m, 2H), 4.33 (t, 1H), 4.02 ( dd, 1H), 3.87 (dd, 1H), 3.77 (t, 2H), 3.58 (t, 2H), 13C NMR (300 MHz, D ² O): 5 = 145.0, 142 , 7, 140.3, 128.2, 99.3, 88.0, 77.8, 69.6, 59.9, 48.5, 41.9. MS: m / z (%): 295.1 (63, [M] +), 163.1 (98), 133.1 (5), 123.1 (88).

N-dimethyl p -NAR:

MS: m / z (%): 283.3 (13, [M] +), 151.2 (100).

Derivatives of 5'-O-acetyl N-alkyl p-NAR (9 ', 10', 11 ', 12', 13 ')

Compound 8 (25 mg, 0.61 jmol) was added at various amine (400 | ^j M) in 0.9 ml of trifluoroethanol. After mixing well, the reaction was stored at 4 ° C overnight. After the reaction was quenched with the addition of HCl to make the pH <7, the organic solvent was removed in vacuo. The residue was dissolved in water and isolated with a disposable Octadecyl-C18 extraction column.

5'-O-acetyl N-methyl p-nicotinic riboside (9 '): 1H NMR (400 MHz, D ² O) 5 = 9.42 (s, H ² , 1H), 9.23 (d, H6, 1H), 8.98 (d,

H ⁴ , 1H), 8.33 (t, H ⁵ , 1H), 6.30 (d, Hr, 1H), 4.73 (t, H ² ', 1H), 4.55 (m, 3H) , 4.40 (t, ³ 'H, 1H), 3.02 (s, 3H), 2.11 (s, 3H).

MS: m / z (%): 311.1 (67, [M] +), 269.1 (13), 221.1 (13), 137.1 (100).

5'-O-acetyl-N-ethyl-p-nicotinic riboside (10 '): MS: m / z (%): 325.1 (12, [M] +), 283.1 (7), 151, 1 (77), 139.1 (22), 115.1

(68).

5'-O-acetyl N-allyl-p-nicotinic riboside (11 '): MS: m / z (%): 337.1 (18, [M] +), 163.0 (71), 138.0 (100), 124.0 (18)

5'-O-ethylenediamine N-methyl-p-nicotinic riboside (12 '): MS: m / z (%): 340.2 (100, [M] +), 298.1 (44), 166.1 (100), 124.0

(82)

5'-O-acetyl N-dimethyl-p-nicotinamide riboside (300 MHz, D ² O) 5 = 9.23 (s, H ² , 1H), 9.19 (d, H6, 1H), 8.78 (d, H ⁴ , 1H),

8.32 (t, H ⁵ , 1H), 6.28 (d, Hr, 1H), 4.72 (m, H ⁴ ', 1H), 4.55 (t, H ² ', 1H), 4 , 52 (d, ⁵ 'H, 2H), 4.40 (t, 3.08 (s, 3H), 2.10 (s, 3H). MS: m / z (%): 325.1 (92 , [M] +), 283.1 (2) 151.1 (100).

Claims (7)

1. A method for preparing nicotinamide riboside of Formula I:

where R1 is amino, comprising the steps of: (i) mix (a) substituted tetraacyl or acyl ribofuranose and (b) a compound of formula II:

wherein R2 is alkyloxy, -OCH ² CH ² OH or aryloxy; to form a compound of formula III:

where R2 is alkyloxy, -OCH ² CH ² OH, or aryloxy, and R3, R4, and R5 are each acyl; and (ii) mixing the compound of formula III with a solution of ammonia to form nicotinamide riboside. 2. The method of claim 1, wherein R2 is phenyloxy. The method of claim 2, wherein step (i) is carried out in the presence of trimethylsilyl trifluoromethanesulfonate. The method of claim 2, wherein the ammonia solution in step (ii) is a solution of ammonia in methanol. 5. A method for preparing nicotinamide riboside, comprising: (i) provide a compound of Formula III:

where R2 is alkyloxy, -OCH ² CH ² OH, or aryloxy, and R3, R4, and R5 are each acyl; and (ii) mixing the compound of formula III with a solution of ammonia to form nicotinamide riboside of formula I.

where R1 is amino. 6. The method of claim 5, wherein R2 is phenyloxy. The method of claim 5, wherein the ammonia solution of step (ii) is a solution of ammonia in methanol.