Technical filed:

The present invention relates to novel process for synthesis of 1,4- dihydronicotinamide riboside (NRH). More particularly, the invention relates to a cost-effective synthesis of 1, 4-dihydronicotinamide riboside (NRH) from Nicotinamide-beta-riboside triacetate chloride (NRA-C1) in good yields and purity.

Background and prior art:

Nicotinamide riboside and derivatives thereof, including nicotinate riboside, nicotinamide mononucleotide, and nicotinate mononucleotide, are metabolites of nicotinamide adenine dinucleotide (NAD+). Nicotinamide adenine dinucleotide (NAD+), and its reduced form, 1,4-dihydronicotinamide adenine dinucleotide (NADH), are the key molecules in energy metabolism and mitochondrial function by electron transfer. Therefore, interest in pharmacological agents capable of increasing cellular NAD+ concentrations has stimulated investigations into nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). However, NR and NMN require large dosages for increasing cellular NAD+ concentrations. Sauve et al. have synthesized 1,4-dihydronicotinamide riboside (NRH) and demonstrated that NRH is a potent NAD+ concentration enhancer in both in vitro and in vivo conditions and further reported that, NRH increases the NAD+ concentration by 2.5-10-fold over control values in just one hour in mammalian cells. Sauve et al. also demonstrated that the use of NRH is more effective than either NR or NMN.

Subsequently, Canto et al. have found that, contrary to the NR pathway, NRH uses different steps and enzymes to synthesize NAD+ and further demonstrated that NRH is orally bioavailable as an NAD+ precursor. Since NRH has gaining importance over NR or NMN, industrially useful synthesis of this molecule is also gaining the attention of research community.

There is ample literature available on the methods for the preparation of NRH.

N. J. Oppenheimer et al reported the synthesis of NRH from dihydronicotinamide mononucleotide (NMNH) by hydrolysis of the 5 '-phosphate ester in the presence of alkaline phosphatase. However, this method is not cost-effective, as it is timeconsuming and involves enzymes. The method of Oppenheimer therefore is not suitable for industrial production of NRH (Biochemistry, 1976, 15, 3981 — 3989).

M. V. Makarov et al (Org. Biomol. Chem., 2019, 17, 8716 — 8720) reported a two-step method for the synthesis of NRH by using triacetylated nicotinamide riboside triflate as a starting material. In the first step, triacetylated NR is converted into triacetylated NRH by reduction with Na2S2O4.In the second step, methanolysis of triacetylated NRH while ball-milling results in the production of NRH. Although this method is promising as it results in good yields of NRH, the use of triacetylated nicotinamide riboside triflate, an expensive and non-food grade substance due to the presence of triflate anion, limits the scalability of this method. The method reported by M. V. Makarov et al is shown in scheme 1 below.

Scheme 1

Y. Yang et al reported another method for the synthesis of NRH by the reduction of NR triflate in the presence of sodium dithionate (Na2S2O4) and potassium hydrogen phosphate as a reducing agent(Biol. Chem., 2019, 294, 9295 — 9307). The crude NRH thus obtained is immediately purified with HPLC using a C18 resin column as NRH is sensitive to both hydrolysis and oxidation at ambient conditions. The method reported by Yang et al provides NRH in a 70% yield. The method is shown in scheme 2 below.

Scheme 2:

0 °C, under argon gas Nicotinamide riboside trifilate Dihydronicotinamide riboside

(NRH)

The disadvantages involved in the above methods are: a) the precursor, nicotinamide riboside triflate, is not only expensive but also very hygroscopic and hence must be stored at about -20 °C under an inert atmosphere, which escalates the additional cost of the production; and b) the nicotinamide riboside triflate is not food grade because of the presence of the triflate anion and hence necessitates the column purification of NRH to completely remove the triflate anions, and hence escalates the cost of the manufacturing process.

Amin Zarei et al (DOI https://doi.org/10.1039/DlRA02062E) reported a direct procedure for the scalable synthesis of NRH by using commercially available P- NRC1. In this method P-NRC1 is reduced in an aqueous solution of NaHCOs and Na2S2O4 under a nitrogen atmosphere. However, this method also has a disadvantage as the resultant product, NRH needs to be purified by a column chromatography from the reaction mixture to obtain the product with 55% yield and 96% purity. Moreover, NRC1 is reported to be a labile molecule that degrades when exposed to elevated temperature which necessitates careful storage and handling. The synthesis reported in this article is shown in scheme 3 below. Scheme 3:

WO2017079195 discloses reaction of triacetylated NR triflate with sodium dithionite to yield the triacetylated NRH, which is deacetylated to yield NRH by treating with a base such as sodium methoxide in MeOH. The process reported in WO’ 195 is also industrially not suitable as triacetylated NR triflate is expensive and not of food grade due to the triflate ion and hence NRH thus obtained needs extensive purification to be free from triflate anion. The synthesis is shown in scheme 4 below.

Scheme 4

The examples 1A and 1C of WO2015/014722 disclose the reaction of triacetylated or tribenzoylated NR triflate with sodium dithionite to yield the triacetylated NRH. Example 2 of WO2015/014722 discloses the deprotection of triacetylated / tribenzoylated NRH with a base such as sodium hydroxide in MeOH under mechanochemical process to yield NRH, as shown in scheme 5 below. This process also requires additional equipment as well as column chromatography purification and hence is not industrially scalable. Scheme 5:

In the light of the foregoing, there remains a need in the art to provide a process for preparation of NRH at least in 85-99% yields and at least with a purity of > 98%, in a cost-effective manner and hence can be commercially scalable.

Therefore, it is an objective of the present invention to provide a process for preparation of 1,4-dihydronicotinamide riboside (NRH) using Nicotinamide-beta- riboside triacetate chloride (NRA-C1), a cost-effective starting material.

Summary of the invention:

In line with the above objective, the present invention provides a process for preparation of 1, 4-dihydronicotinamide riboside (NRH), comprising the steps of; a) reducing Nicotinamide-beta-riboside triacetate chloride (NRA-C1) in presence of non-chlorinated aprotic organic solvent(s) and a reducing agent to obtain 1, 4-dihydronicotinamide riboside triacetate (NRH-A); and b) deacetylating the 1 ,4-dihydronicotinamide riboside triacetate (NRH- A) in presence of a base to obtain 1 ,4-dihydronicotinamide riboside (NRH).

In an aspect, the reducing agent may be selected from the group consisting of sodium dithionite (Na2S2O4) or Thiourea dioxide (TDO).

The non-chlorinated aprotic organic solvents for the reduction of NRA-C1 can be selected from the group consisting of cyclic and acyclic ethers such as 2- methyltetrahydrofuran (2-MeTHF), t-butylmethyl ether (TBME), di isopropyl ether, diethyl ether, cyclo pentyl methyl ether, esters such as ethyl acetate (EtO Ac), methyl acetate, propyl acetate, butyl acetates and cyclic and acyclic hydrocarbon solvents such as cyclohexane, cycloheptane, n-hexane, n-heptane, benzene, xylenes, trimethylbenzenes and toluene. In particular, water insoluble solvents are preferred as the reaction takes place in biphasic solvent system.

The base which can be used for deacetylation of NRH-Ais selected from the group consisting of 0.1% NaOH in MeOH, 0.1% NaOMe in MeOH, 1 mol% K2CO3 in methanol, 7% methanolic NH3 and 10% aqueous NH3 or di alkyl amines such as N,N-dimethyl or diethyl or dibutyl amine or cyclic secondary amines such as pyrrolidine, morpholine and piperidine in protic solvents like methanol, ethanol and isopropanol.

Description of drawings:

Fig 1 depicts 1HNMR of NRH-OAc, as prepared in accordance with the example 2.

Fig 2 depicts 13C NMR- NRH-OAc as prepared in accordance with the example 2.

Fig 3 shows 1H NMR of NRH prepared in accordance with the example 3.

Fig 4 shows 13C NMR- NRH prepared in accordance with the example 3.

Fig 5 shows HPEC chromatogram of NRH prepared in accordance with the example 3. Figure 6 shows Mass spectrum of NRH prepared in accordance with the example 3.

Figure 7 shows PXRD of NRH prepared in accordance with the example 3 indicating the amorphous nature of the compound.

Figure 8 shows HRMS of NRH prepared in accordance with the example 3.

Detailed description:

The invention will be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be fully understood and appreciated.

Accordingly, the present invention provides a process for preparation of 1 ,4- dihydronicotinamide riboside (NRH), comprising the steps of; a) reducing Nicotinamide-beta-riboside triacetate chloride (NRA-Cl) in presence of non-chlorinated aprotic organic solvent(s) and a reducing agent to obtain 1 ,4-dihydronicotinamide riboside triacetate (NRH- A); and b) deacetylating the 1 ,4-dihydronicotinamide riboside triacetate (NRH- A) in presence of a base to obtain 1 ,4-dihydronicotinamide riboside (NRH).

According to an embodiment, reduction of Nicotinamide-beta-riboside triacetate chloride (NRA-Cl) was carried out using sodium dithionite (Na S CE) in presence of a saturated solution of NaHCOsundcr nitrogen atmosphere in presence of a suitable non-chlorinated organic solvent.

The non-chlorinated aprotic organic solvents for the reduction of NRA-Cl can be selected from the group consisting of cyclic and acyclic ethers such as 2- methyltetrahydrofuran (2-MeTHF), t-butylmethyl ether (TBME), di isopropyl ether, diethyl ether, cyclo pentyl methyl ether, esters such as ethyl acetate (EtO Ac), methyl acetate, propyl acetate, butyl acetates and cyclic and acyclic hydrocarbon solvents such as cyclohexane, cycloheptane, n-hexane, n-heptane, benzene, xylenes, trimethylbenzenes and toluene. More particularly, the non-chlorinated organic solvents for this reaction can be selected from the group consisting of 2-methyl tetrahydrofuran (2-MeTHF), t- butylmethyl ether (TBME), ethyl acetate (EtOAc), cyclohexane and toluene. The reduction reaction can be carried out suitably from room temperature to the reflux temperature of the solvent used. Preferably, the reduction reaction was carried out at room temperature under stirring for 4-5 h. After the completion of the reaction, the product, 1,4-dihydronicotinamide riboside triacetate(NRH-A) was recovered from the organic phase.

In one of the preferred aspects, the suitable non-chlorinated organic solvents are selected from 2-methyl THF or ethyl acetate. 2-methyl THF is particularly a useful solvent among various ethers, as the same is a green solvent, also recoverable and reusable and further gave the product in quantitative yield with excellent purity. Thus, the present invention demonstrated the conversion of NRA-C1 into NRH-A which is devoid of the use of chlorinated solvents such as DCM, and DCE.

Reduction of nicotinamide-beta-riboside triacetate chloride may take place in a yield of >70% in toluene, >75% in diethyl ether, >80% in tert-butyl methyl ether, >95% in ethyl acetate, or >90% in 2-methylTHF. Reduction of nicotinamide- betariboside triacetate chloride may produce 1 ,4-dihydronicotinamide riboside triacetate with a purity of >85% in toluene, >90% cyclohexane, >98% in 2- methylTHF or ethyl acetate. Further, the NRH-A thus obtained was subjected to deacetylation in presence of a suitable base at room temperature. Upon complete disappearance of starting material as indicated by TEC (2:8 MeOH:DCM), the solvent was evaporated and the thick mass thus obtained was washed twice with acetone and then dried under vacuum over 1-2 h to afford light yellow solid, NRH. The base for this deacetylation reaction can be selected from the group consisting of 0.1% NaOH in MeOH, 0.1% NaOMe in MeOH, 1 mol% K2CO3 in methanol, 7% methanolic NH3 and 10% aqueous NH3. In one of the preferred embodiments, the base is 0.1% sodium methoxide in methanol.

Deacetylation of 1,4-dihydronicotinamide riboside triacetate may take place in a yield of >80% by ammonia, >85% by K2CO3, >90% by NaOH or >96%NaOMe in methanol. Deacetylation of 1,4-dihydronicotinamide riboside triacetate may produce NRHwith a purity of >90% by K2CO3, >95%NaOMe, >98%NaOH.

In another preferred embodiment, reduction of Nicotinamide-beta-riboside triacetate chloride (NRA-C1) was carried out using Thiourea dioxide in presence of a suitable non-chlorinated aprotic organic solvent in saturated, solution of NaHCCh, as a base under nitrogen atmosphere. The reduction reaction can be carried out suitably from room temperature to the reflux temperature of the solvent used; however, the reduction reaction was preferably carried out at room temperature under stirring for 6-8 h. After the completion of the reaction, the product, NRH-A was recovered from the organic phase. The non-chlorinated aprotic organic solvents which can be used for this reaction, including: cyclic and acyclic ethers, e.g., diethyl ether, diisopropyl ether, THF, 2- methyltetrahydrofuran, and t-butylmethyl ether(TBME); cyclopentylmethyl ether esters, e.g., C1-C6 alkyl esters of C1-C6 carboxylic acids, such as ethyl acetate, methyl acetate, propyl acetate and butyl acetate;

Cl -CIO cyclic alkanes; e.g., cyclohexane, cycloheptane

C1-C10 acyclic alkanes; e.g., n-hexane, n-heptane and aromatic hydrocarbons, e.g., benzene, toluene, xylenes, and trimethylbenzenes .

In various embodiments, exemplary non-halogenated aprotic organic solvents which can be used for reduction of nicotinamide-beta-riboside triacetate chloride include 2-methyltetrahydrofuran (2-MeTHF), t-butylmethyl ether (TBME), ethyl acetate (EtOAc), cyclohexane and toluene.

Thiourea dioxide is a readily available reducing reagent for the selective 1,4- reduction of NRA-C1 under extremely mild conditions. In one of the preferred embodiments, the reaction was carried out in 2-methyl THF solvent, which gave the product in good yield with excellent purity.

Further, the NRH-A thus obtained was subjected to deacetylation in presence of a suitable base at room temperature. Upon complete disappearance of starting material as indicated by TLC (2:8 MeOH:DCM), the solvent was evaporated and the thick mass was washed twice with acetone and then dried under vacuum over 1-2 h to afford light yellow solid, NRH. The base that can be employed for this deacetylation reaction is selected from the group consisting of 0.1% NaOH in MeOH, 0.1% NaOMe in MeOH, 1 mol% K2CO3 in methanol, 7% methanolic NH3, 10% aqueous NH andanhydrous HC1 in methanol.

In one of the preferred embodiments, the base is 0.1% sodium methoxide in methanol.

The starting material, P-NRCl-OAc, as used in the present invention for the synthesis of NRH is stable at room temperature and hence, handling of this material is easier and require no special conditions for storage. Further, the use of P-NRCl-OAc provides the NRH-A in 85-95% yields as well as NRH in 90-95% yields and 90-98% purity compared to the reported processes. The process of the present invention facilitates the separation of the intermediate, NRH-A as well as the final product NRH easily from the organic phase, due to the use of judicial selection of solvents, without subjecting to column chromatography purifications unlike the teachings of prior arts. Moreover, the entire process can be conducted at room temperature thereby avoiding requirement for additional source of energy. Therefore, the process of the present invention for the synthesis of NRH is cost- effective and hence is industrially scalable.

The invention will now be illustrated with help of examples. The aforementioned embodiments and below mentioned examples are for illustrative purpose and are not meant to limit the scope of the invention. Various modifications of aforementioned embodiments and below mentioned examples are readily apparent to a person skilled in the art.

Examples

Example 1

Process for synthesis of NRH-A from NRA-C1 using Na S tL in 2-methyl

THE

Stage 1:

Reduction of NRA-C1 using sodium dithionite (Na2S2O4)

NRA-C1 (2.8 g, 7.4 mmol) was dissolved in 40 mL of nitrogen purged 2-methyl THF under N2 atmosphere were added sat. solution of NaHCOs (13 mL) and solid sodium dithionite (4.17 g, 24 mmol) followed by 5 mL of water at 25 °C. The resulting mixture was stirred at the same temperature over 4-5 h. The progress was monitored by TLC using 1% methanol in ethyl acetate or 1% methanol in DCM as a mobile phase and TLC spots were visualized by anisaldehyde solution. The organic phase was separated and the aqueous phase was extracted with 40 mL of 2-methyl THF. The combined organic layers were washed with brine solution, dried over sodium sulphate and concentrated under reduced pressure to afford the pale-yellow solid (NRH-A) in 90% yield with > 98% purity.

Example 2

Process for synthesis of NRH from NRH-A using Na S tb in ethyl acetate

Stage 1: Reduction of NRA-C1 using sodium dithionite (Na2S2O4)

Into a three neck 3L RB flask fitted with a magnetic stirrer were charged Ethyl acetate (1400 mL) and nitrogen was purged for 30 min and added NRA-C1 (100 g) and the contents were stirred for 10 min. To this solution, added saturated Sodium bicarbonate solution (464 mL) (RM turns to a light yellow colour) followed by the addition of sodium dithionite (solid, 148.9g) slowly under stirring at 25 °C (reaction mixture turns to pale yellow turbid solution). Added DM water (250 mL) slowly to get the clear solution, the contents were stirred at room temperature for 2h and monitored by TLC (5% methanol: 95% DCM, TLC spots were visualized by anisaldehyde solution or 10% H2SO4 Methanol solution. After the completion of the reaction, separated the organic layer, washed with brine solution (500mL), dried over Na2SO4 and distilled out the organic layer under reduced pressure maintaining the bath temperature below 39-40°C to obtain a pale yellow solid product in 88g (95.8% yield).

1HNMR and 13C NMR of NRH-OAc, are shown in figures 1 and 2 respectively.

Similarly, the reduction of NRA-C1 using sodium dithionite (Na2S2O4) was conducted in various other solvents such as t-butylmethyl ether (TBME), cyclohexane and toluene. As shown in below table 1, the reduction of NRA-C1 using sodium dithionite (Na2S2O4) produces 90% yield in 2-Methyl THF;95.8% yield in ethylacetate; 75% in t-Butylmethyl ether(TBME); 70% in cyclohexane; 65% in Toluene. Further, all these solvents produce the product, NRA-H, with a purity of 85% to 98%. Also, the reaction is carried out at a temperature of 25 to 30°C. Table 1: a ^,b Deacetylation was observed along with the reduction and purification was tedious. cReaction was carried out at 40 °C

All of these solvents were proved to be suitable as these solvents results in NRH- A with good yields and therefore, are useful for the industrial production of NRH-

A. Stage 2:

Deacetylation of NRH-A (2) using sodium methoxide in methanol

To a stirred solution of 2 (80gm) obtained by the process of above examples 1 and 2 in methanol (400ml) was added sodium methoxide (1.15 g) at 25 °C. The resulting solution was stirred at the same temperature over 3-4h. Upon complete disappearance of starting material as indicated by TLC (2:8 MeOH:DCM). Distilled the organic layer under reduced pressure maintaining the bath temperature below39-40°C to obtain a pale yellow solid product in 52g (97 % yield).

Purification:

The pale yellow solid product NRH (50g) was dissolved in 500ml acetone and stirred vigorously for 30min. After 30min, filter the product under strict nitrogen atmosphere and washed with acetone 250ml. The product filtered was dried under nitrogen atmosphere to obtain amorphous powder.

Purity: 99.97% by HPLC Shimadzu with Lab solutions software

1H NMR, 13CNMR, HPLC chromatogram, Mass spectrum, PXRD and HRMS of NRH are provided in figures 3 to 8 respectively.

Similarly, the deacetylation reaction was also earned out using different bases such as 0.1% NaOH in MeOH, 1 mol% K2CO3 in methanol, 7% methanolic NH3 and 10% aqueous NHs.and the product NRH was obtained in very good yields indicating the suitability of these bases for industrial scalability of the reaction.

Example 3:

Process for synthesis of NRH from NRA-C1 using Thiourea dioxide (TDO) in 2-MeTHF

Stage 1:

NRA-C1 (2.8 g, 7.4 mmol) was dissolved in 40 mL of nitrogen purged 2-methyl THF under N2 atmosphere were added sat. solution of NaHCOs (13 mL) and solid TDO (1.59 g, 14.8 mmol) at 25 °C. The resulting mixture was stirred at the same temperature over 6-8h. The organic phase was separated and the aqueous phase was extracted with 40 mL 2-methyl THF. The combined organic layers were washed with brine solution, dried over sodium sulphate and concentrated under reduced pressure to afford the pale-yellow solid (NRH-A) in 82% yield with > 98% purity.

Similarly, the reduction of NRA-C1 using Thiourea dioxide (TDO) was conducted in various solvents that result in a yield of 75% in t-butylmethyl ether (TBME); 80% in ethyl acetate (EtOAc); 70% in cyclohexane; and 65% in toluene. All of these solvents were proved to be suitable, as these solvents results in NRH-A with good yields and purity (table 1), for the industrial production of NRH-A. Similarly, the reduction reaction was carried out using different bases which produces NRH-A in a yield of 85% by aqueous NH3; 80% by alcoholic NH3; 65% by KaHPCUand 60% by K3PO4. However, the acetate hydrolysis was also observed simultaneously with aqueous NH3 or alcoholic NH3. Aqueous NH3 or alcoholic NHsdcprotccts acetate groups along with reduction of NRA-C1.

Stage 2:

Deacetylation of NRH-A (2) using sodium methoxide

To a stirred solution of 2 (80g) in methanol (400ml) was added sodium methoxide (1.15g) at 25 °C. The resulting solution was stirred at the same temperature over 3-4h. Upon complete disappearance of starting material as indicated by TLC (2:8 MeOH:DCM), distilled the organic layer under reduced pressure maintaining the bath temperature below 39-40°C to obtain a pale yellow solid product in 52g (97 % yield).

Purification:

The pale yellow solid product NRH (50g) was dissolved in 500ml acetone (lOvol) and stirred vigorously for 30min. After 30min, filter the product under strict nitrogen atmosphere and washed with acetone 250ml (5vol). The product filtered is dried under nitrogen atmosphere and transferred to flame dried nitrogen flushed vial.

Purity: 99.97% by HPLC Shimadzu with Lab solutions software Similarly, the deacetylation reaction was carried out using different bases which produces NRH in a yield of >95% by 0.1% NaOH or NaOMe in MeOH; 85% by 1 mol% K2CO3 in methanol; 90% by 7% methanolic NH3 and 87% bylO% aqueous NH3, indicating the suitability of these bases and the scalability of this reaction.

Industrial applicability:

The starting material, P-NRCl-OAc, is stable at room temperature and hence, handling of this material is easier and requires no special conditions for storage.

Further, the use of P-NRCl-OAc provides the NRH-A as well as NRH in good yields and purity compared to the reported processes.

The process of the present invention facilitates the separation of the intermediate, NRH-A as well as the final product NRH easily from the organic phase due to the use of judicial selection of solvents, without subjecting to column chromatography purifications unlike the teachings of prior arts. The process can be carried out at room temperature and pressure and thus avoids the requirement of an energy source for conducting the reaction. The NRH obtained by the process of the present invention is amorphous in nature and is of high purity (>99.97% by HPLC).

Therefore, the process of the present invention for the synthesis of NRH is cost- effective and hence is industrially scalable.