Deracemization of Axially Chiral Nicotinamides by Dynamic Salt Formation with Enantiopure Dibenzoyltartaric Acid (DBTA)

Dynamic atroposelective resolution of chiral salts derived from oily racemic nicotinamides and enantiopure dibenzoyltartaric acid (DBTA) was achieved by crystallization. The absolute structures of the axial chiral nicotinamides were determined by X-ray structural analysis. The chirality could be controlled by the selection of enantiopure DBTA as a chiral auxiliary. The axial chirality generated by dynamic salt formation was retained for a long period after dissolving the chiral salt in solution even after removal of the chiral acid. The rate of racemization of nicotinamides could be controlled based on the temperature and solvent properties, and that of the salts was prolonged compared to free nicotinamides, as the molecular structure of the pyridinium ion in the salts was different from that of acid-free nicotinamides.

Spontaneous resolution by crystallization is a powerful tool for obtaining optically active axially chiral materials [34][35][36][37][38][39]; we recently reported a new example of spontaneous chiral symmetry breaking of an axially chiral nicotinamide, obtaining the product in up to 94% enantiomeric excess (Scheme 1) [40]. The nicotinamide shown in Scheme 1 afforded a conglomerate, and each enantiomer formed enantiomorphic crystals. When the amide was crystallized from the melt in the first racemization, based on C-C(=O) bond rotation, a high ee of crystals was obtained with good reproducibility. Total optical resolution of racemic nicotinamide was achieved by a combination of preferential crystallization and racemization. Scheme 1. Deracemization of racemic nicotinamide 1 by spontaneous chiral crystallization from the melt.
Optically active materials can be conveniently obtained by this spontaneous chiral resolution; however, the fine methodology involving crystallization is not applicable to all crystalline materials, but is successful only in conglomerate crystals [34]. It is known that conglomerate crystals are formed at a rate of only 10% in organic racemic materials [41,42]. In most cases optically active materials crystallize in chiral fashion. Racemic mixtures may be converted to diastereomers through the formation of chiral salts with enantiopure materials followed by optical resolution by preferential crystallization [43,44]. Dynamic preferential crystallization is particularly useful for amino acids and pharmaceutical reagents [45,46].
We examined the deracemization of nicotinamides by salt formation with enantiopure acids, and succeeded in controlling their axial chirality by deracemization through the growth of crystalline salts. Finally, the chiral acid was removed and a chiral memory of free nicotinamides with axial chirality was generated. To our knowledge, there have been no previous examples of control of axially chiral nicotinamides by salt formation.

Results and Discussion
Deracemization of oily racemic nicotinamides with a basic group was examined by dynamic salt formation with enantiopure acids such as dibenzoyltartaric acid (DBTA), based on the fact that chiral materials tend to crystallize in chiral fashion [34,47,48]. Dynamic racemization through the formation of crystalline salts achieves effective deracemization of the racemic base to give optically active salts. For example, if an enantiopure (R)-acid is added to the racemic base under conditions of efficient racemization, and the crystallinity of the (S)-base/(R)-acid salt is superior to that of the other diastereomeric salt [(R)-base/(R)-acid], effective dynamic optical resolution can be achieved and the (S)-base/(R)-acid form will be obtained in enantiopure form with good chemical yield (Scheme 2). Furthermore, the chiral acidic adjuvant can easily be removed by extraction under basic conditions. This is a convenient method of obtaining axially chiral materials. The use of an enantiomeric acid such as an (S)-acid gives enantiomeric chiral salts with an (R)-base through deracemization in an easy-touse approach.

Scheme 2. Deracemization by dynamic salt formation.
To examine the deracemization of axially chiral nicotinamides, salt formation was carried out using three oily derivatives, 1a-c, with enantiopure DBTA (Figure 1). When racemic 1a and an equimolar amount of L-DBTA were dissolved in chloroform and the solvent was removed by an evaporator, a viscous oil was obtained. To analyze the ee of 1a and determine whether deracemization was promoted by salt formation, the salts were dissolved in chloroform and washed with aqueous NaHCO 3 to remove acidic DBTA. The ee value of free 1a was determined by HPLC using a Daicel CHIRALPAK ® AD-H column (Scheme 3). Deracemization was not observed when the materials were mixed without crystallization ( Table 1, entry 1). In the cases of 1b and 1c, removal of the solvent by evaporation led to a viscous oil, and deracemization was not observed (entries 3 and 6). In previous experiments involving deracemization by salt formation in the solution phase, it was demonstrated that external chiral stimuli induced a helical sense in a dramatically optically active helical polymer or oligomer [49][50][51][52][53][54]. In our experiment, the axial chirality of 1a-c could not be controlled in the solution phase because the chiral acid in the salt was located far from the axial chiral auxiliary. In contrast, all of the amides formed crystalline salts with enantiopure DBTA by slow evaporation of the solvent. A chloroform solution of racemic 1a and an equimolar amount of enantiopure L-DBTA in a test tube was warmed to 60 °C to accelerate the deracemization of 1a, and removal of the solvent led to the formation of crystalline salts. After all of the solvent had been slowly evaporated off, crystalline salts remained at the bottom of the test tube. The process took about 12 h in all cases. After the removal of DBTA by extraction, the ee of 1a was analyzed by HPLC ( Figure 2). The upper part of the figure shows the trace by the UV detector, while the lower trace is that obtained from the CD detector. Five experiments on salt formation of 1a with L-DBTA gave optically active 1a in ee of 60% to 67% with (−) specific optical rotation (entry 2). This result indicates that deracemization was promoted by dynamic crystalline salt formation. Table 1. Deracemization of racemic nicotinamides 1a-c by salt formation with (−)-L-DBTA.

Entry
Amide Notes: a Ee was determined by HPLC using Daicel CHIRALPAK ® AD-H column after DBTA was removed by extraction using aqueous NaHCO 3 ; b All nicotinamides 1a-c obtained by the use of L-DBTA showed (−) sign of specific optical rotation. Absolute conformation was established by X-ray analysis; c Racemic 1 and equimolar amount of L-DBTA was mixed and the solvent was removed by evaporator for each five experiments; d Crystallization was examined each five times. Each 100 mg of 1a-c and equimolar amount of L-DBTA was used for salt formation. Nicotinamides were recovered quantitatively without loss after salt formation under these conditions; e Racemic 1b and equimolar amount of D-DBTA was used for dynamic salt formation; f Absolute configuration was not determined; however, the 1a after removing L-DBTA showed the (−) specific rotation. In the case of deracemization of 1b by salt formation with L-DBTA, a better ee was obtained (from 75% to 83% ee) in five experiments (entry 4). In these experiments, (−)-1b was also obtained after removal of L-DBTA, as in the case of 1a, and the ee value was determined by HPLC ( Figure 3). When enantiopure (+)-D-DBTA was used for salt formation, (+)-1b was obtained with almost the same ee value (entry 5). Both enantiomers of 1b could be easily prepared by selection of the appropriate enantiomeric DBTA. Deracemization of 1c by salt formation with L-DBTA was also successful, and a 51%-60% ee of 1c was obtained (entry 6), which also showed (−) specific optical rotation, after removal of DBTA. Racemization of 1c was slightly faster than that of 1a and 1b, as described below. HPLC analysis was performed at 0 °C ( Figure 4). Acid-free nicotinamide 1c was prepared by extraction of the obtained salt from aqueous NaHCO 3 ; therefore, the ee value may have decreased during the workup process. Recrystallization from acetone or CHCl 3 gave enantiopure single crystals for X-ray structural analysis for all salts except that of 1a/L-DBTA. X-ray analysis of the chiral salts of 1b and L-DBTA recrystallization from acetone revealed the absolute conformation and the molecular arrangement, which was an orthorhombic crystal system with space group P2 1 2 1 2 1 .  Figure 5 shows the perspective view. The crystal contained two acetone molecules beside 1b and L-DBTA. Protonation occurred on the nitrogen atom of the pyridine ring, and a near-planar conformation was revealed between the pyridinium ring and the pyrrolidine group at the 2-position. It is known that 2-amino nicotinamides have a distorted conformation between the pyridine ring and the cyclic amino group at the 2-position [40]. These facts indicated that the protonated salts had a different molecular conformation from that of acid-free 1, and suggest strong conjugation of the lone pair electrons of the nitrogen atom of the pyrrolidine ring with the pyridinium function. In contrast to the planar conformation of the pyrrolidine ring at the 2-position, the amide plane was almost perpendicular to the pyridine ring (dihedral angle: 80.1°). The absolute conformation of the axial chirality of (−)-1b was an (S)-conformation. The consistency of the absolute conformation of 1b in the single crystal and the major diastereomeric salt was certified by the HPLC analysis.      Recrystallization of chiral salts of 1c and L-DBTA from CHCl 3 gave enantiopure single crystals available for X-ray structural analysis. A disordered structure was observed in the morpholine and pyrrolidine groups (Figure 9). Protonation occurred on the nitrogen atom of the pyridine ring, and the pyrrolidine ring on the 2-position adopted an almost planar conformation against the pyridine ring. Furthermore, the absolute conformation of axial chirality was (S), which was the same as that of the salt of 1b and L-DBTA. The consistency of the absolute conformation of 1c in the single crystal and the major diastereomeric salt was certified by the HPLC analysis and the comparison of the experimental powder diffraction pattern with the calculated powder pattern from single crystal data. Figure 10 shows the packing diagram; a 2 1 helix is shown along with the b-axis. Two kinds of intermolecular hydrogen bond were observed: (a) between NH (pyridinium ion) and O(O=)C (DBTA), with a distance of 1.95 Å, and (b) between two DBTA units (distance 1.68 Å).
For efficient chiral symmetry breaking, fast racemization is required in the crystallization process. In contrast, for practical use of the generated axial chirality in subsequent asymmetric reactions, chiral recognition, etc., chirality should be retained for a long period by slow racemization. Thus, efficient deracemization can have opposing requirements. If the rate of racemization is too low, deracemization cannot be achieved. When it occurs, effective deracemization can be achieved under fast racemization conditions; however, axial chirality may be lost as soon as the salt is dissolved in the solvent. The rate constants (k rac ) and activation parameters for racemization were examined to estimate the conformational stability of 1a-c in three types of solvent-CHCl 3 , MeCN, and methanol. To analyze the racemization of acid-free 1a, chiral salt 1a/L-DBTA obtained by deracemization was dissolved in CHCl 3 , and DBTA was removed by extraction with aqueous NaHCO 3 . The rate of racemization was determined based on changes in the specific optical rotation at 20 °C or 30 °C. The same method was used for 1b and 1c. The activation free energies and half-lives are listed in Table 2.   because of the most planar pyrrolidine ring. In methanol (a protic and polar solvent), the rate of racemization was lowest in all cases (entries 3, 6, and 10). In the case of 1a, the rate was measured at 30 °C (entry 3), because racemization was too slow to measure at 20 °C. As shown in entries 7 and 11, salts showed slower racemization than acid-free nicotinamides. This is reasonably explicable on the basis of their conformation. Acid-free 2-alkylaminonicotinamides have a conformation that is twisted between the pyridine ring and the cyclic amino group at the 2-position [40]. However, X-ray analysis (shown in Figures 5, 7 and 9) showed that the pyridinium ion conjugates with the lone pair of electrons of the nitrogen atom at the 2-position. The planar conformation of the cyclic amino group at the 2-position prevents the easy rotation of the amide function at the 3-position.
These findings indicate that the chirality may be controlled by varying the temperature and solvent properties. At high temperatures bond rotation can occur, resulting in fast racemization of these materials, and they may be deracemized to optically active axially chiral nicotinamides by salt formation with enantiopure acid. Furthermore, racemization was suppressed around room temperature, and chirality could be retained for a long period even after removal of the chiral auxiliary DBTA.
Furthermore, a protic solvent such as methanol strongly inhibited racemization; polarity, hydrogen bonding, and solvation by alcohol were important factors that influence the rate of racemization. The effects of solvent polarity may be attributable to the zwitterionic character of the amide group, and solvation of the hydrogen bond with protic solvents reduces the rate of bond rotation.

General
NMR spectra were recorded in CDCl 3 solution on a Bruker 300 instrument operating at 300 MHz for 1 H-and 13 C-NMR spectroscopy. Chemical shifts are reported in parts per million (ppm) relative to TMS as an internal standard. IR spectra were recorded on a JASCO FT/IR-230 spectrometer. Specific rotation was measured using a DIP 370 polarimeter (JASCO). X-ray single crystallographic analysis was conducted using a SMART APEX II or SMART APEX II ULTRA (Bruker AXS).

Preparation of Nicotinamides 1a-c
To a toluene solution containing 2-chloro-4,6-dimethylnicotinic acid (2.00 g, 10.8 mmol) [55] was added thionyl chloride (1.90 g, 16.2 mmol), and the mixture was refluxed for 6 h at 80 °C. After removal of the solvent and excess thionyl chloride in vacuo, crude 2-chloro-4,6-dimethylnicotinyl chloride was obtained and used for the subsequent reaction. To a toluene solution of the crude 2-chloro-4,6-dimethylnicotinyl chloride, pyrrolidine (1.83 g, 27.0 mmol) was added dropwise at 0 °C. After the reaction mixture had been stirred for 2 h at room temperature, water and ethyl acetate were added, and the organic layer was extracted in the usual manner. After evaporation of the organic solvent in vacuo, the residual mixture was subjected to chromatography on silica gel, and N-(2-chloro-4,6-dimethyl-3-pyridinecarbonyl)pyrrolidine was separated in 80% yield. After N-(2-chloro-4,6dimethyl-3-pyridinecarbonyl)pyrrolidine (1.00 g, 4.2 mmol) had been refluxed with pyrrolidine (3.57 g, 42.0 mmol) in an argon atmosphere overnight, water and ethyl acetate were added and the organic layer was extracted in the usual manner. The residual mixture was subjected to chromatography on silica gel and the corresponding nicotinamide 1a was separated in 70% yield. Other nicotinamides 1b-c were prepared in the same manner. Structures were determined on the basis of spectral data.

Deracemization of Nicotinamides 1a-c by Crystalline Salt Formation
One hundred mg aliquots of 1 were used for salt formation. A CHCl 3 solution of racemic nicotinamide 1 and equimolar amount of enantiopure L-or D-DBTA in a test tube was warmed up to 60 °C with stirring until all solvent was slowly evaporated off. Then the crystalline salts remained at the bottom of the test tube, it took about 12 h in all cases.

Determination of the ee Value of 1 after Removing Chiral Acid
After DBTA was removed by extraction, the ee of 1 was analyzed by HPLC. Chiral salts were dissolved to a cooled mixture of CHCl 3 and aq. NaHCO 3 , and the organic layer was separated and washed with cooled water. And the CHCl 3 solution containing free nicotinamides was analyzed by HPLC using a CHIRALCEL-ADH column.

Kinetic Studies for Racemization of 1
The rate of racemization of 1 was studied in three types of solvent: CHCl 3 , MeCN and methanol. Optically active 1 was prepared from the corresponding chiral salt. The chiral salt was dissolved in CHCl 3 and the solution was washed with cooled aqueous NaHCO 3 , water and brine. After the organic layer had been dried with anhydrous MgSO 4 , the solvent was evaporated in vacuo at room temperature. Changes in the specific rotation of the crude nicotinamides in the three types of solution were monitored at 20 °C or 30 °C. The activation parameters were obtained from the Eyring equation. The first-order kinetic plots of the decay profile of the ee values were shown as a plot of ln(ee) versus time [Equation (1)], and the rate of racemization (k rac ) was calculated from the slope of the line. The free energy barrier (ΔG ‡ ) for racemization was calculated based on the Eyring equation [Equation (2)]. The half-life was calculated based on Equation (3): ln(ee) = k rac t (1) k rac = (kT/h)exp(−ΔG ‡ /RT) (2) t 1/2 = ln2/2k rac (3) where k rac : rate of racemization, h: Planck constant, k: Boltzmann constant, R: gas constant, T: temperature.

Conclusions
We have provided a fine example of symmetry breaking to give axial chirality in nicotinamides through the formation of crystalline salts with enantiopure DBTA, which promoted effective dynamic deracemization. The absolute structure of the axial chiral compound became clear, and it was possible to obtain the desired axially asymmetric compound by the selection of enantiopure DBTA as a chiral auxiliary. Furthermore, a kinetic study of racemization showed clearly that the chiral conformation was retained for a considerable time even after removal of the chiral acid. Furthermore, the rate of racemization of nicotinamides could be controlled by selection of the temperature and solvent properties, and that of the salts was prolonged compared to free nicotinamides because the molecular structure of the pyridinium ion in the salts was different from that of acid-free nicotinamides.
Nicotinamides are well known not only as NAD/NADH model systems, but also as catalysts for many asymmetric reactions. The axially chiral nicotinamides obtained in this work are expected to be useful for subsequent asymmetric reactions and precursors of various types of optically active heterocycle.