Polymorphism of Nicotinamide Riboside Chloride and Crystal Structure Determination of the Most Stable Crystalline Form

Three crystalline forms of nicotinamide riboside chloride (NR-Cl), namely Form A, Form B and Form C, were prepared and characterized. Form A and Form B are true polymorphs of anhydrous forms, while Form C is a pseudo-polymorph of a methanolate solvate form. The relative polymorphic stability relationship among these three crystalline forms was established, and Form B was found to be the most stable form with a higher crystal density (1.591 Mg/m3) and a higher melting/decomposing temperature (123 °C) compared to that of Form A (1.512 Mg/m3 and 119 °C). The crystal structure of Form B (orthorhombic P212121 space group, a = 7.0008(11) Å, b = 9.6465(15) Å, c = 17.971(3) Å, V = 1213.7(3) Å3, Z = 4) was determined by single crystal X-ray diffraction analysis. The crystal structure of the most stable polymorph of nicotinamide riboside chloride (NR-Cl) is reported. The crystal structure of the most stable polymorph of nicotinamide riboside chloride (NR-Cl) is reported.

Nicotinamide riboside is marketed as nicotinamide riboside chloride salt (NR-Cl) with a chemical structure shown in Fig. 1. Preparation methods of various crystalline forms of NR-Cl were disclosed in a number of patents in recent years [4][5][6][7][8][9], and these crystalline forms are believed to have advantageous physicochemical properties compared to the amorphous form, e.g. higher chemical purity and better physical stability.Crystalline Form A recrystallized from H 2 O/EtOH mixed solvents was first discovered by GlaxoS-mithKline [4], and the same crystalline form was also identified from MeOH/TBME mixed solvents by W. R. Grace & Co. at the same time [5].The second crystalline Form B recrystallized from MeOH/Acetone/H 2 O mixed solvents was disclosed by W. R. Grace & Co. [6].Both Form A and Form B were confirmed as anhydrous forms.Form C was identified as a methanolate solvent from MeOH solvent [7].
The single crystal growth by the vapor diffusion method and crystal structure determination of Form A ((orthorhombic P2 1 2 1 2 1 space group, a = 4.9373(1) Å, b = 7.4170(1) Å, c = 34.8797(5)Å, V = 1277.29(4)Å 3 , Z = 4) by single crystal X-ray crystallography were also reported recently [10].However, the preparation and polymorphism relationship among all disclosed crystalline forms were not systematically investigated.Herein, we report the preparation of three crystalline forms, the relative polymorphic stability relationship among these forms, and the crystal structure determination of the most stable polymorph of Form B of nicotinamide riboside chloride (NR-Cl).

Experimental Section
Nicotinamide riboside chloride (NR-Cl) (Form B) was purchased from UltraHealth LLC (727 Central Avenue, Worthington, IN 47471, USA), and all crystallization solvents were used without further purifications.

Preparation of NR-Cl-Form A
A clear colorless solution of NR-Cl was prepared by dissolving 200 mg of white solid nicotinamide riboside chloride (NR-Cl) in 0.5 mL of water (H 2 O).4.0 mL of ethanol (EtOH) was added, resulted in a cloudy solution.4.0 mL of tert-butyl methyl ether (TBME) was added slowly to the cloudy solution to precipitate white solid, and then the slurry was stirred at the room temperature for 2 h.170 mg of white solid was obtained after the homogenous slurry was filtered and dried in vacuum for 4 h (yield: 85%).XRD confirmed that the solid is NR-Cl-Form A, whose XRD pattern is shown in the bottom graph of Fig. 2.

Preparation of NR-Cl-Form B
A clear colorless solution of NR-Cl was prepared by dissolving 500 mg of white solid nicotinamide riboside chloride (NR-Cl) in 2.5 mL of water (H 2 O).5.0 mL of acetone and 2.5 mL of isopropanol (IPA) were added to the solution causing a white solid to precipitate.The suspension was stirred at room temperature for 1 day and a homogenous slurry was formed.385 mg of white solid NR-Cl-Form B was obtained after the homogenous slurry was filtered and dried in vacuum for 4 h (yield: 77%).XRD confirmed that the solid is NR-Cl-Form B, whose XRD pattern is shown in the middle graph of Fig. 2.
Single crystals of NR-Cl-Form B were grown from evaporation of a MeOH/dichloromethane (DCM) solution (or IPA/H 2 O solution) of NR-Cl with NR-Cl-Form B seeds.

Preparation of NR-Cl-Form C
A clear colorless solution of NR-Cl was prepared by dissolving 50 mg of white solid nicotinamide riboside chloride (NR-Cl) in 1.0 mL of methanol (MeOH).2.0 mL of tertbutyl methyl ether (TBME) was added to the solution causing a white solid to precipitate.The suspension was stirred at the room temperature for 1 day and a homogenous slurry was formed.22 mg of white solid from was obtained after the homogenous slurry was filtered and dried in vacuum for 4 h (yield: 44%).XRD confirmed that the solid is NR-Cl-Form C, whose XRD pattern is shown in the upper graph of Fig. 2.

Competitive Slurry Study of NR-Cl-Form A and NR-Cl-Form B
20 mg of white solid NR-Cl-Form A and 20 mg of NR-Cl-Form B were suspended in 0.50 mL of ethanol or 0.50 mL of acetone.The slurry was stirred at the room temperature.XRD showed that a mixture of Form A and Form B converted a pure Form B after 5 days in the EtOH slurry, and it also showed a mixture of Form A and Form B converted to a pure Form B after 8 days in the acetone slurry.The XRD patterns of the competitive slurry studies are shown in Supplementary information S4.

Slurry Study of NR-Cl-Form C
20 mg of white solid NR-Cl-Form C was suspended in 0.50 mL of acetone.The slurry was stirred at room temperature for 1 day.XRD showed that NR-Cl-Form C was converted to NR-Cl-Form A, as shown in Supplementary information S5(b).

X-Ray Powder Diffraction (XRPD) Analysis
XRPD analysis was performed using a Bruker D8 diffractometer, Bruker AXS Inc™ (Madison, Wisconsin).The XRPD spectrum of each sample was obtained by mounting a sample (approximately 5-10 mg) on a single silicon crystal wafer mount (e.g., a Bruker silicon zero background X-ray diffraction sample holder) and spreading out the sample into a thin layer with the aid of a microscope slide.The sample was spun at 30 revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a copper long-fine focus tube operated at 40 kV and 40 mA with a wavelength of 1.54 Å.Each sample was exposed for 1 s per 0.02° 2θ increment (continuous scan mode) over the range of 5° to 40° degrees 2θ in θ-θ mode.

Differential Scanning Calorimetry (DSC) Analysis
DSC analysis was performed on samples prepared according to standard methods using a Q SERIES™ Q1000 DSC calorimeter available from TA INSTRUMENTS® (New Castle, Delaware).A sample (approximately 2-5 mg) was weighed into an aluminum sample pan and transferred to the DSC.The instrument was purged with nitrogen into the sample and reference compartment at 50 mL/min and data were collected between 22 and 300 °C, using a dynamic heating rate of 10 °C/minute.Thermal data was analyzed using Universal v.4.5A from TA INSTRUMENTS®.

Thermogravimetry Analysis (TGA)
TGA was performed on samples prepared according to standard methods using a Q SERIES™ Q5000 thermogravimetry analyzer available from TA INSTRUMENTS® (New Castle, Delaware).A sample (approximately 5-8 mg) was placed into an aluminum sample pan and transferred to the TGA furnace.The instrument was purged with nitrogen at 50 mL/min and data were collected between 25 and 300 °C, using a dynamic heating rate of 10 °C/minute.Thermal data was analyzed using Universal v.4.5A from TA INSTRUMENTS®.

Single Crystal Analysis
Single crystal analysis was performed using a Bruker Apex diffractometer, Bruker AXS Inc™ (Madison, Wisconsin).The diffraction data were collected at 23 °C using Mo X-ray source.The crystal structure was solved and refined with the SHELXTL package [15].The H atoms on the N and O atoms were located in the electronic density map while all other H atoms on the C atoms were calculated.The four restraints imposed during the refinement came from the restraints for two hydrogen atoms (H3O and H5O bonded with O3 and O5) that were located from difference Fourier map and refined with proper (necessary) restraints.
The simulated X-ray powder pattern of NR-Cl-Form B was calculated from the single crystal structure data with the help of program Mercury [16] from The Cambridge Crystallographic Data Centre (CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336,033).
It is worth mentioning that a ternary solvent system with two mixed anti-solvents were used in preparation of both NR-Cl-Form A and NR-Cl-Form B. In preparation of NR-Cl-Form A, the single anti-solvent of ethanol only precipitated a small amount of the product.Yield was significantly improved when using the second anti-solvent of TBME.In this case, the ethanol not only serves as an anti-solvent but also overcomes miscibility issue between water and TBME.In preparation of NR-Cl-Form B, a ternary solvent system is necessary for crystallization of NR-Cl-Form B. It was observed that water/acetone binary solvents only produced a gel-like solid, while water/isopropanol binary solvents gave a low yield of the product, which was often found as a mixture of NB-Cl-Form A and NR-Cl-Form B.

Solid State Characterization of NR-Cl Crystalline Forms
The overlay of powder X-ray diffraction (XRD) patterns of NR-Cl-Form A, NR-Cl-Form B and NR-Cl-Form C is shown in Fig. 2. While Form A and Form B demonstrated high crystallinity, Form C had some background noise, indicating that the material contained some content of amorphous form and was likely not very stable due to de-solvation of methanol solvate.Some caution should be taken in the XRD characterization of Form C since the conversion of Form C to Form A was observed when Form C was pressured with a microscope slide while being flattered on the XRD sample holder.XRD patterns prior to and after compressing the Form C sample on the XRD sample holder were shown in Supplementary information S5(a), showing partial conversion of Form C to Form A. Such an observation of form conversion in the XRD measurement correlates with the crystalline solvate form which was disclosed in GlaxoSmithKline's patent WO2015186068 [4].This solvate form had an XRD pattern comparable to that of Form A, even though the solvate form itself contained about 0.9 molar ratio of methanol in the material.Likely, this was due to the form conversion during XRD measurement.More studies are needed to confirm this hypothesis.
The overlay of differential scanning calorimetry (DSC) curves of NR-Cl-Form A, NR-Cl-Form B and NR-Cl-Form C is shown in Fig. 3.The first endothermal event for Form A and Form B was melting of crystalline form and chemical decomposition of the nicotinamide riboside into nicotinamide and sugar, confirmed by a previous study [11].The followed exothermal event was assumed to be crystallization of the nicotinamide compound, and the XRD pattern of the crystalline form obtained after heating Form A or Form B in a TGA pan matched with that of the crystalline form of the nicotinamide [11].The endothermic event of Form B with an onset temperature of 123 °C revealed a higher

Patent Summary and Nomenclature of Crystalline Forms
Three crystalline forms of NR-Cl have been discovered so far and disclosed in various patents as listed in Table 1.Table 1 also designates these forms into an ambiguous nomenclature.The crystalline form nomenclature is based on observation of XRPD pattern similarity, and the figures of XRPD overlays of each crystalline form are shown in supplementary information.
Three distinguishable crystalline forms were observed in the previous patents and crystalline forms are designated as Form A, Form B and Form C, respectively.

Preparation of Crystalline Forms
Each of the three crystalline forms was prepared by dissolving NR-Cl into a solvent, e.g.H 2 O or MeOH, followed by addition of the anti-solvent(s), in which NR-Cl has a poor solubility, to precipitate the desired crystalline form.The resulting suspension was stirred at the room temperature to yield a homogenous slurry and then isolated by filtration.XRD was utilized for the crystalline form identification.
White solid NR-Cl is readily dissolved in H 2 O or MeOH to obtain a clear colorless solution.NR-Cl shows a better solubility in water compared to MeOH so that water is preferred to dissolve white solid NR-Cl, except for NR-Form C, which is a methanolate solvate and requires methanol during crystallization.Interestingly, both polymorphs of NR-Cl-Form A and NR-Cl-Form B were identified by addition of different anti-solvents into the aqueous solution.data are shown in Table 2 and the content of the asymmetric unit and crystal packing is shown in Fig. 5.The absolute configuration of the enantiomerically pure molecule was confirmed by the Flack parameter (0.02(1)) and matched with the expected stereochemistry.NR-Cl-Form B crystallizes in an orthorhombic P2 1 2 1 2 1 space group, the same space group with NR-Cl-Form A, which was determined previously [10].NR-Cl cations in Form A and Form B adopted different molecular conformations, and comformational polymorphs between Form A and Form B were confirmed, as shown in Fig. 6.The torsional angle connecting nicotinamide and sugar shows about 180° rotation (The torsional angle of C1-N1-C7-O2 is 34.6(4)° in Form B vs. -136.1(1)° in Form A), consequentially the amide group of nicotinamide lies towards the same side of the sugar group in Form B but at the opposite side in Form A. Besides, the hydroxyl groups in the riboside group are in different directions.As a consequence, NR-Cl-Form B reveals unique hydrogen bonding and crystal packing in crystal lattices compared to those of NR-Cl-Form A. For example, both hydrogen atoms in the amide group in Form B bonded to the chloride anion, However, only one of the two hydrogen atoms in the amide group of Form A bonded to the chloride anion while the other one bonded to the amide melting/decomposing temperature compared to that of Form A with an onset temperature of 119 °C, suggesting Form B is more thermally stable than Form A. The first endothermic event of Form C with an onset temperature of 79 °C was assumed to be a de-solvation process of this methanolate solvate form.
The overlay of thermal gravimetric analysis (TGA) curves of NR-Cl-Form A, NR-Cl-Form B and NR-Cl-Form C is shown in Fig. 3.The TGA indicates that both Form A and Form B exhibit an insignificant weight loss (< 0.5%) in the solvent region (25-105 °C), confirming that Form A and Form B are anhydrous crystalline forms, and true polymorphs.Form C exhibits a weight loss of about 7.5% upon heating from 25 °C to about 105 °C, close to the methanol content for a mono-methanolate form (The theoretical methanol content of mono-methanolate is 32.04/ (290.70 + 32.04) = 9.9%).

Crystal Structure of NR-Cl-Form B
Single crystals of NR-Cl-Form B were obtained from slow evaporation of the NR-Cl solution.It should be noted that seeding with NR-Cl-Form B in the saturated solution is critical to obtain the desired Form B crystals.Otherwise, Crystalline NR-Cl-Form A was obtained.The crystallographic   intermolecular hydrogen bonding is listed in Table 3.

Relative Polymorphic Stability of NR-Cl Crystalline Forms
Polymorph studies aim to identify as many as possible crystalline forms and establish relative polymorphic stability relationship among these forms.As a result, it helps to ensure the optimal crystalline form, usually the most stable form, is selected for commercial product development.Form A and Form B are true polymorphs of anhydrous forms, while Form C is a pseudo-polymorph of the solvated form.The solvated crystalline form is usually not preferred due to its potential physical stability issue and the possible toxicity caused by the extra solvent content.Thus it becomes critical to establish the polymorphic stability relationship between the two true polymorphs, Form A and Form B. A quick competitive slurry study demonstrated that a mixture of Form A and Form B converted to pure Form B at ambient temperature, which suggests that NR-Cl-Form B is more stable than NR-Cl-Form A. Such a conclusion from the competitive slurry is also supported from the studies of thermal analysis and single crystal analysis, and the most stable Form B has higher melting/decomposing temperature (123 °C of Form B vs. 119 °C of Form A) and higher crystal density (1.591 Mg/m 3 of Form B vs. 1.512Mg/m 3 of Form As shown in Fig. 7, the overlay of the simulated powder X-ray diffraction pattern calculated from single crystal structure data matches well with the experimental XRD pattern of NR-Cl-Form B. The XRD identicalness not only indicates that the characterized crystal structure represents NR-Cl-Form B but also confirms that the prepared bulk material is a pure crystalline form of NR-Cl-Form B.  A), thus Form B is preferred to be used in pharmaceutical or nutraceutical development.
It is also worth pointing out that NR-Cl-Form B, as the most physically stable form, is also expected to possess a better chemical stability.Crystalline form control is crucial in order to produce the desired NR-Cl-Form B with the optimal physical and chemical stability, which would benefit the shelf life of a commercial product.It would be especially valuable to investigate the improvement of the chemical stability and the shelf life by using the most stable polymorph of Form B. It should be kept in mind that the experimental polymorph study itself does not guarantee that any identified crystalline form is the most stable.There is always an opportunity to discover a more stable form during further crystallization experiments.While ongoing crystallization study continues to identify novel crystalline form of NR-Cl, other substitute products, including new crystalline NR organic salts [12], NR prodrugs [13] and nicotinamide mononucleotide (NMN) as an alternative NAD + precursor [14], have also been under investigations in order to achieve optimal physicochemical properties.

Conclusions
In summary, three crystalline forms of NR-Cl were prepared and characterized.NR-Cl-Form A and NR-Cl-Form B are two polymorphs of anhydrous form, while NR-Cl-Form C is a methanolate solvate.Competitive slurry, thermal analysis and single crystal analysis studies conclude that NR-Cl-Form B is more stable compared to NR-Cl-Form A at ambient temperature, suggesting that NR-Cl-Form B is preferred in pharmaceutical or nutraceutical substance development.The single crystal structure of NR-Cl-Form B reveals molecular conformation and crystal packing of this most stable polymorph at the molecular level.

Fig. 2 X
Fig. 2 X-ray powder diffraction patterns of crystalline forms of NR-Cl.

Fig. 4
Fig. 4 Overlay of TGA curves of crystalline forms of NR-Cl.

Fig. 3
Fig. 3 Overlay of DSC curves of crystalline forms of NR-Cl.

Fig. 7
Fig. 7 Overlay of the simulated XRD and the experimental XRD of NR-Cl-Form B