Green and Efficient Resolution of Racemic Ofloxacin using Tartaric

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Green and Efficient Resolution of Racemic Ofloxacin using Tartaric Acid Derivatives via Forming Co-crystal in Aqueous Solution Lichao He, Zhongrui Liang, Guojia Yu, Xiangrong Li, Xinjian Chen, Zhiyong Zhou, and Zhongqi Ren Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00414 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Crystal Growth & Design

Green and Efficient Resolution of Racemic Ofloxacin using Tartaric Acid Derivatives via Forming Co-crystal in Aqueous Solution Lichao He, Zhongrui Liang, Guojia Yu, Xiangrong Li, Xinjian Chen, Zhiyong Zhou* and Zhongqi Ren* College of Chemical Engineering, Beijing University Chemical Technology, NO. 15, N. 3rd Ring Rd East, Beijing 100029, People’s Republic of China

ABSTRACT: The separation of chiral compounds into their enantiomers is widely used in the pharmaceutical industry. Two types of environmentally benign tartaric acid

derivatives,

O,

O′-dibenzoyl-(2S,3S)-tartaric

acid

(D-DBTA)

and

O,

O′-dibenzoyl-(2R,3R)-tartaric acid (L-DBTA), were selected as efficient chiral selectors for the separation of racemic ofloxacin by forming diastereomeric co-crystal pairs in the aqueous phase. Effects of type of chiral selectors, resolution time, the amounts of racemic ofloxacin, and temperature on the resolution performance of ofloxacin were investigated. The results indicated that D-DBTA selectively co-crystallized with the R-enantiomer (R-OFLX), while L-DBTA selectively co-crystallized with S-enantiomer (S-OFLX) in the aqueous phase. Under the optimal conditions, the enantiomer excesses (% ee) reached up to 81.8% ee and 82.3% ee for S-OFLX and R-OFLX at 278.2 K, respectively. The stoichiometric ratio of D-DBTA or L-DBTA to R-ofloxacin or S-ofloxacin in co-crystals was 1:1. Various characterization and calculation methods, such as Powder X-ray Diffraction (PXRD), Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), and Density Functional Theory (DFT),

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were performed for the study of the resolution mechanism, the results of which demonstrated that the co-crystals were formed by hydrogen bonding between D-DBTA or L-DBTA and ofloxacin. Compared with previous studies, the proposed separation process was not only environmentally benign but also beneficial to increasing % ee. The proposed method has explored a new path for green and efficient separation of racemic compounds which can’t form salts. KEYWORDS: Chiral separation, Ofloxacin, Green and efficient, Co-crystal, DBTA INTRODUCTION Separation of racemic compound into single enantiomer is an important process, not only for the pharmaceutical and fine chemical industries,1,2 but also for the agrochemical and food industries.3 Over the past decades, the development of efficient methods to obtain chiral compounds in enantiopure form has attracted increasing attention. The target single enantiomer can be obtained via two distinctive pathways of asymmetric synthesis4 and chiral resolution. Though asymmetric synthesis of pure enantiomers has incredible advantages,5-8 it needs long development time and high development cost. The chiral separation approach relies on selectively removing one enantiomer from a racemate via various technologies, such as enantioselective liquid-liquid extraction,9 electrophoresis,10 enzymatic kinetic resolution,11 ion-exchange,12 membrane-based technology13 and molecular imprinting technology.14,15 However, these resolution techniques are either too slow or too expensive, which is not beneficial to large sale application.

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To date, the formation of diastereomeric salt16-21 and chiral chromatography22 are two common industrially applied techniques for chiral resolution. For those compounds that can’t or can’t easily form salts, chiral chromatography seems to be the only viable technique for enantioseparation in industrial production in spite of expensive cost. On this point, chiral separation by forming co-crystal, most relying on intermolecular hydrogen bonding formed between active pharmaceutical ingredient (API) and co-crystal former, becomes an interesting alternative method for the compounds which can’t or can’t easily form salts. In recent years, pharmaceutical co-crystals studies have attracted increasing attention. It is well known that pharmaceutical co-crystals exhibit excellently biopharmaceutical properties, such as dissolution rate, solubility, hygroscopicity, stability, and etc.23-27 The enantioselective co-crystallization has already been investigated for chiral resolution.28-30 Eddleston et al.31 studied the formation of diastereomeric co-crystals between malic acid and tartaric acid by liquid-assisted grinding method. However, the results only indicated that the formation of co-crystal by grinding could be a potential way for chiral resolution. Springuel et al.32 prepared the enantiospecific co-crystals of butanamide with S-mandelic acid. 52.66% ee was obtained in acetonitrile at -10 oC. These results suggest that the chiral separation of racemic compounds can be realized by co-crystallization. However, rare investigations on the chiral separation by co-crystallization have been reported so far. Thus, it is urgent to develop a totally environmentally benign and efficient method for enantioseparation by co-crystal for those compounds that can’t form salts.

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In this study, in order to show the great application potential of this technique, racemic ofloxacin which can’t easily form salts and can’t be separated efficiently using traditional chiral resolution techniques was selected as model pharmaceutical compound. Ofloxacin is an important fluoroquinolone antibiotic and the drug activity of the S-enantiomer (levofloxacin) is 8-128 times higher than that of the R-enantiomer. As shown in Figure 1, racemic ofloxacin could be separated by the tartaric acid derivatives via forming co-crystal in aqueous solution. In this work, the influence of the structure of chiral selectors on ofloxacin resolution efficiency was investigated. Additionally, effects of experimental conditions, such as the resolution time, the amounts of chiral selectors and racemic ofloxacin and temperature on resolution performances were also studied. Finally, the mechanism of chiral resolution was studied by various characterization methods like XRD, TG, DSC, FT-IR and DFT calculations. This study may provide important information for finding green and efficient separation process for racemic compounds which can’t or can’t easily form salts. EXPERIMENTAL SECTION Reagents and Materials. Both S-ofloxacin (S-OFLX, C18H20FN3O4, purity > 99%) and racemic ofloxacin (OFLX, C18H20FN3O4, purity > 99%) were purchased from Beijing Innochem technology Co., Ltd. Beijing, China. Methanol (CH4O, chromatographic grade) was purchased from Tianjin Shield Specialty Chemicals Co., Ltd., Tianjin, China. D-DBTA (C18H14O8, purity > 99%), L-DBTA (C18H14O8, purity > 99%), O,O′-dibenzoyl-(2S,3S)-4-toluoyl-tartaric acid (D-DTTA, C20H18O8, purity >

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99%), O,O′-dibenzoyl-(2R,3R)-4-toluoyl-tartaric acid (L-DTTA, C20H18O8, purity > 99%), Di-(+)-p-methoxy-D-tartaric acid (D-DMTA, C20H18O10, purity > 99%) and Di-(-)-p-methoxy-L-tartaric acid (L-DMTA, C20H18O10, purity > 99%) were supplied by Beijing HWRK Chem Co., Ltd., Beijing, China. Anhydrous copper sulfate (CuSO4, purity > 99%) was purchased by Beijing Chemical Works, Beijing, China. L-Leucine (C6H13NO2, purity > 99%) was purchased by Aladdin Industrial Corporation, Shanghai, China. All the chemicals were used as received without any further purification. The water used in all experiments was prepared by filtration through an ultrapure purification system (Shanghai SKYLARK Industry Co., Ltd., Shanghai, China). Characterization. Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectra have been recorded on a 8700 attenuated total refection Fourier transform infrared spectrometer (Nicolet, Fisher Scientific, USA) in the range from 500 cm−1 to 4000 cm−1. Thermogravimetric Analysis (TGA). TGA measurements were carried out on a TG thermal analyzer (TG209C, Netzsch, Germany) in a dynamic N2 atmosphere. Approximate 10 mg samples were placed into aluminum pans and analyzed in the temperature ranging from 35 °C to 800 °C with a heating rate of 10 °C·min-1. Differential Scanning Calorimetry (DSC). DSC measurements were carried out on a differential scanning calorimeter (PERKIN ELMER, USA). Prior to measurements, the differential scanning calorimeter was calibrated using indium. The heating rate was 10 °C·min-1. The temperature ranged from 25 °C to 180 °C.

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Powder X-ray Diffraction (PXRD). The PXRD measurements were carried out on a Rigaku Ultima IV X-ray Powder diffractometer (Tokyo, Japan) at 40 kV, 100 mA with a Cu Kα radiation (λ = 1.5406 Å). PXRD patterns were recorded from 5° to 40° in 2θ with a scan rate of 1°·min-1. Equilibrium solubility study. The solubilities of racemic ofloxacin, tartaric acid derivatives and co-crystals in aqueous solution were determined. An excess amount of the solid phase was added to 20 mL deionized water in a flask, and the solution was continuously stirred at temperature of 5, 10, 15, 25, 35 or 40 °C for 24 h to reach equilibrium. Then the solution was filtered through a 0.45-µm nylon filter and analyzed quantitatively by high performance liquid chromatography (HPLC). Separation Procedures. The separation experiments were carried out in 50 mL conical flasks with cover. Equal volume (25 mL) of ofloxaicn aqueous solution was added in the conical flask, followed by adding an appropriate amount of chiral selectors. The mixture was shaken sufficiently in a water bath at a certain temperature and then left to equilibrate and settle for 5 min to ensure that the solid-liquid equilibrium reached. The solid-liquid mixture could be separated by centrifugation and filtration. At last, the concentrations of ofloxacin enantiomers in aqueous solution were analyzed quantitatively by HPLC. Sample Analysis. HPLC analysis method for OFLX. The chiral HPLC analysis was performed on a LC-20AT high performance liquid chromatograph (Shimadzu, Japan) equipped with an UV (SPD-20AT) detector and Zorbax Extend C18 column (length of 250 mm, internal diameter of 4.6 mm, particle size of 5µm) purchased from

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Agilent. The injection volume was 10 µL and column temperature was maintained at 39 °C. A mixed solution (50% ultrapure water, 50% methanol, v/v) with flow rate of 1 mL·min-1 was used for elution. The mobile phase consisted of methanol and aqueous solution (85:15, v/v) containing 10 mmol·L-1 L-leucine and 5 mmol·L-1 CuSO4, which was employed at a flow rate of 0.8 mL·min-1. The effluent was monitored at 295 nm. The measured retention times for S-ofloxacin and R-ofloxacin were 15.5 min and 17.5 min, respectively. The relevant liquid chromatographic diagrams for the separation and detection of ofloxacin stereomers are shown in Supporting Information. HPLC analysis method for DBTA. The mobile phase consisted of methanol, ultrapure water, and glacial acetic acid (60:40:1, v/v), which was employed at a flow rate of 0.5 mL·min-1 at 231 nm. The measured retention times for both D-DBTA and L-DBTA were 10.5 min. The other parameters were the same as HPLC analysis method for OFLX. The distribution coefficient, kS and kR, separation factor, α, and enantiomeric excess value, % ee, can be obtained by the following equations,

kS =

kR =

α=

ee =

CS,S

(1)

C L,S CS,R

(2)

CL,R

kS kR

or

CL,S − CL,R CL,S + CL,R

α=

× 100%

kR kS or

(3)

ee =

CL,R − CL,S CL,R + CL,S

× 100%

7


(4)


where CL,S and CS,S represent concentrations of S-ofloxacin in the liquid phase and solid phase after separation, respectively. CL,R and CS,R represent concentrations of R-ofloxacin in the liquid phase and solid phase after separation, respectively. The CL,S (g·L-1) and CL,R (g·L-1) values were determined by chiral HPLC analysis. Moreover, the CS,S (g·L-1) and CS,R (g·L-1) values were calculated by material balance.

Density Functional Theory (DFT) calculation. The Materials Studio DMol3 program was used for all the calculations in this study. The atom valence orbitals were described by double numerical plus polarization (DNP) basis set, which has been stuied by Delley.33 The Becke-Lee-Yang-Parr (BLYP) functional of the generalized gradient approximation (GGA) was used to obtain the nonlocal exchange and correlation energies. The convergence criteria consisted of threshold values for energy, force, and displacement convergence were 1×10-5 hartree, 0.002 hartree·Å-1, and 0.005 Å, respectively. All the geometry optimizations were carried out at this level, and and the properties of electron density, frequency and electrostatics were considered during the calculations. Determination of Crystal structure from PXRD. The crystal structure was predicted from PXRD by the Material Studio Reflex program. There are four steps in the overall prediction process, such as indexing, pawley fitting, structure solution and rietveld refinement.34 In the indexing step, the crystal class and the approximate lattice parameters could be obtained from the peak positions in the experimental PXRD patterns using TREOR. Based on their figure of merit, a table could be obtained from the results of TREOR, which arranged the proposed unit cells. The unit 8


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cell with the highest figure of merit was selected to generate an empty unit cell. The agreement of the calculated powder pattens with the experimental powder patterns was confirmed by the Rwp (weighted Rietveld parameter) value obtained by Pawley fitting, which also ensured the accurate crystallographic parameters. Then, the optimized drug molecular struture was added into the refined unit cell and motion groups were defined. The Reflex Powder Solve module was used to obtain the structure with the Monte Carlo/simulated annealing procedure. There were 10 cycles of simulated annealing and each cycle involved 200,000 steps. Finally, the final structure solution and Rwp value were obtained by Rietveld refinement. RESULTS AND DISCUSSION Effect of Tartaric Acid Derivatives Type on Resolution Performance. L-DBTA, D-DBTA, L-DTTA, D-DTTA, L-DMTA and D-DMTA were selected as typical tartaric acid derivatives for investigation of the effect of chiral selector type on resolution performance. The formation of co-crystal depends on the molecular interaction between tartaric acid derivative and ofloxacin. Due to various spatial structures of the selected typical tartaric acid derivatives, the chiral selectors may show different enantioseparation performances towards OFLX enantiomers. As shown in Figure 2, D-DBTA, L-DTTA and D-DMTA show stronger recognition abilities for R-OFLX than S-OFLX. Meanwhile, the other three derivatives show reversed recognition abilities. It can be seen that the separation factor and % ee for racemic ofloxacin in the aqueous phase with D-DBTA and L-DBTA as chiral selectors are much higher than that with the other four chiral selectors. the maximum % 9



ee for S-enantiomer in the aqueous phase is 79.3% ee with 0.08 g D-DBTA as chiral selector, while the maximum % ee for R-enantiomer in the aqueous phase is 78.2 % ee with 0.08 g L-DBTA as chiral selector. Therefore, D-DBTA and L-DBTA were determined as preferred chiral selectors in aqueous solution. Effect of Resolution Time on Resolution Performance. As shown in Figure 3, effect of resolution time on resolution performance was investigated in this study. Both the separation factor and % ee value for R-ofloxacin and S-ofloxacin first increase sharply with the increase of resolution time within 30 min and then increase slightly from 30 min to 50 min. Both the resolution equilibriums for R-ofloxacin and S-ofloxacin were reached at 50 min. The results of preliminary experiments showed that R-ofloxacin preferentially interacted with D-DBTA, which is beneficial to the existence of R-ofloxacin in the solid phase. By contrast, S-ofloxacin was preferentially transferred into the solid phase with L-DBTA as chiral selector, due to the different formation abilities of molecular interaction. The maximum % ee values for S-ofloxacin and R-ofloxacin in the aqueous phase are 79.3% ee with D-DBTA as chiral selector and 78.2% ee with L-DBTA as chiral selector at 50 min, respectively. This short equilibrium time indicates that there is a relatively rapid mass transfer for the formation of co-crystal between DBTA and oflxoacin. Therefore, in the following work, 50 min was selected as optimal resolution time. Effect of Chiral Selectors Amount on Resolution Performance. In chiral liquid-solid process, the separation enantioselectivity is also influenced by the addition amount of chiral selectors. As shown in Figure 4, effect of the amount of 10


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chiral selectors on resolution performance was studied. When the addition amount of D-DBTA or L-DBTA is less than 0.02 g, almost no enantioselectivity for ofloxacin could be obtained with rare formation amount of diastereomeric co-crystals between DBTA and ofloxacin. However, when the addition amount of D-DBTA or L-DBTA reaches approximately 0.03 g, a large number of solids began to appear. The higher the addition amount of DBTA, the stronger the ability of forming co-crystals, which is beneficial to the increase of the separation enantioselectivity for racemic ofloxacin. Both separation factor and % ee values for ofloxacin increase with increasing the addition amount of DBTA from 0.03 g to 0.08 g. However, both separation factor and % ee values for ofloxacin almost have no change with increasing the addition amount of DBTA from 0.08 g to 0.10 g. Once the addition amount of DBTA is higher than 0.08 g, the stability difference between diastereomeric co-crystal pairs will become unobvious and the % ee value of ofloxacin will reach a plateau. The maximum % ee value of S-ofloxacin in the liquid phase was 79.3% ee with 0.08 g D-DBTA as chiral selector and the maximum % ee value of R-ofloxacin in the liquid phase was 78.2% ee with 0.08 g L-DBTA as chiral selector. Therefore, 0.08 g was selected as the suitable addition amount of chiral selectors. Effect of Racemic Ofloxacin Amount on Resolution Performance. The resolution performance with D-DBTA as chiral selector depends on the solubility difference of a diastereomeric co-crystal pairs consisting of R-OFLX:D-DBTA and S-OFLX:D-DBTA in the aqueous phase. As shown in Figure 5, the separation factor and % ee value for OFLX in the aqueous solution increase gradually with increasing 11



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OFLX concentration. The higher the ofloxaicn concentration is, the stronger the intermolecular interaction between host and guest molecules is. This can be due to the fact that most separations of two enantiomers at high concentration are through enantioselective co-crystallization. When the concentration of OFLX is higher than 1.6 g·L-1, the ofloxacin would not be completely dissolved. Therefore, 1.6 g·L-1 was selected as the optimal concentration of OFLX. Effect of Temperature on Resolution Performance. The standard enthalpy change (∆H°), entropy change (∆S°) and Gibb’s energy change (∆G°) were obtained by Eqs. (5) and (6).

ln K =

−∆H o ∆So + RT R

(5)

∆Go = ∆H o − T ∆S o

(6)

where R is the gas constant (8.314 J·mol-1·K-1), T is the absolute temperature (Kelvin). As shown in Figure 6 and Figure 7, effect of temperature on resolution performance was investigated. Both separation factors and % ee values for R-OFLX and S-OFLX decrease slightly with increasing temperature. Therefore, the solubility difference between these two co-crystals and the recognition ability of DBTA decrease slightly with increasing temperature. However, both the yields of R-OFLX and S-OFLX in the aqueous phase increase with increasing temperature, indicating that both the solubilities of DBTA:R-OFLX co-crystal and DBTA:S-OFLX co-crystal increase with the increase of temperature. As stated above, the co-crystals are formed 12


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by hydrogen bonding between ofloxacin and DBTA. The hydrogen bonding is much stronger than the molecular interaction between ofloxacin and H2O, leading to the formation of co-crystals. In consideration of separation efficiency and energy consumption, 308.2 K would be suitable for the separation process. The ∆H° and ∆S° values for the resolution process with DBTA as chiral selectors could be calculated from the linear regressions of ln K vs. 1/T via the Van’t Hoff equation. As shown in Figure 8 and Table 1, all the values of R2 are higher than 0.98, which indicates that the experimental data fit very well with Van’t Hoff equation. The values of enthalpy change (∆H°) are negative, indicating that the formation of co-crystals between ofloxacin and DBTA are driven by a exothermic change. Thus, decreasing the temperature is beneficial to co-crystallization. The negative values of entropy change (∆S°) obtained in this study represent a decrease in the degree of freedom at the solid-liquid interface.35 In addition, the negative charge of Gibb’s free energy (∆G ° ) values suggest that the selective co-crystallization process is spontaneous. Moreover, the obtained absolute value of ∆G°(KJ/mol) for R-OFLX is higher than that for S-OFLX with D-DBTA as chiral selector, indicating that D-DBTA preferentially recognizes R-enantiomer in the aqueous phase. The opposite conclusion can be obtained for L-DBTA. The Determination of Mole Ratio of D-DBTA or L-DBTA to Oflxoacin in Co-crystals by Slurry Crystallization. In order to determine the accurate composition of the co-crystals, all the co-crystals were prepared based on a reported synthetic method.36 Slurry crystallization experiments were conducted with a certain 13



amount of ofloxacin and tartaric acid derivatives for the preparation of co-crystals. For instance, a solution of S-ofloxacin was prepared by dissolving approximately 25 mg S-ofloxacin in 25 mL water in a flask. In another flask, a solution of D-DBTA which was near saturation was prepared by dissolving approximately 50.2 mg D-DBTA in 25 mL deionized water. Then the D-DBTA solution was added to the beaker containing S-ofloxaicn. The co-crystallization reaction was carried out by the sufficient stirring with magnetic stirrer at room temperature. After about an hour, a large amount of co-crystals appeared by the slurry crystallization. The solid phase obtained by filtration was vacuum-dried and then the samples were characterized by XRD, TG, DSC and FT-IR Spectroscopy. Moreover, a solid-liquid equilibrium analysis based on the mass balance was performed to determine the mole ratio of D-DBTA or L-DBTA to oflxoacin in co-crystals. The results listed in Table 2 show that 1:1 D-DBTA:S-OFLX, 1:1 D-DBTA:RS-OFLX, 1:1 L-DBTA:S-OFLX and 1:1 L-DBTA:RS-OFLX co-crystals were formed in slurry crystallization experiments, indicating that the stoichiometric ratio of D-DBTA or L-DBTA to R-ofloxacin in co-crystals is also 1:1. The Resolution Mechanism for Racemic Ofloxacin with DBTA as Chiral Selector. In this study, computational studies were used to speculate on the formation of the co-crystal between D-DBTA and S-OFLX. The molecular electrostatic potential (MEP) plays a vital role in explaining the effect of the electron density distribution (EDD) on physical outcomes in the system, such as atom reactivity and weak interaction.37,38 Thus, the charge density studies of D-DBTA and S-OFLX were 14


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carried out to rationalize the physical properties obtained from the co-crystal structure by charge density analysis. The MEP maps (Figure 9) were mapped onto an isosurface of ρ and plotted on the same scale for comparability. Visual analysis of the MEP showed that C=O and –OH of the carboxyl group in D-DBTA or S-ofloxacin molecule had the maximum negative value and maximum positive value, respectively. Due to high level of complementarity between electronegative and electropositive regions, the hydrogen bond between D-DBTA and S-ofloxacin could be formed by attraction interaction between the positive and negative static electricities. In accordance to the previous findings mentioned above, the carboxyl group on the molecular structure of ofloxacin prefers to form hydrogen bond with the functional group -COO in D-DBTA or L-DBTA, leading to the formation of co-crystals. Based on the formation mechanism of co-crystal reported by Zhang et al.39 and the results stated above, a resolution mechanism was proposed to explain the resolution procedure in which D-DBTA and L-DBTA formed diastereomeric co-crystal pairs with two enantiomers of ofloxacin, which is beneficial to designing for new co-crystals. FT-IR is commonly used to analyze how hydrogen bonding is participated in the reaction process.40 The FT-IR spectra of ofloxacin, D-DBTA, L-DBTA, and co-crystals are shown in Figure 10. Taking FT-IR spectra of S-ofloxacin, D-DBTA, and co-crystals formed by S-ofloxacin and D-DBTA (Figure 10a) as an example, the carbonyl group stretching vibration at 1724.25 cm-1 in S-ofloxacin and 1727.45 cm-1 in D-DBTA shifted to 1718.46 cm-1 in the co-crystal. The electron donating group – 15



COO- transferred partial the electron cloud of the oxygen atoms during the formation of hydrogen bonding, resulting in the change of vibrational state of C=O of ofloxacin molecule in co-crystal.41 The stretching vibration of v-OH could be another criterion of the hydrogen bonding force. The characteristic peak at 3430.68 cm-1 corresponding to the stretching vibration of v-OH in S-ofloxacin shifted to 3437.72 cm-1 in D-DBTA:S-OFLX co-crystal, while shifted to 3438.70 cm-1 in L-DBTA:S-OFLX co-crystal. In this work, the infrared vibration frequency for hydrogen bonding formation between D-DBTA and S-OFLX was calculated based on DFT using the Materials Studio DMol3 program from Accelry. The calculation details were described in the Experimental Section. As shown in Figures S5-S7 in Supporting Information, it can be found that the –OH in D-DBTA tends to give proton to form hydrogen bond with C=O in S-OFLX, leading to decreasing the electron density of C=O, which results in the red-shift of v(C=O) in co-crystal D-DBTA:S-OFLX. Both the C=O stretching vibration peaks in S-ofloxacin at 1741 cm-1 and in D-DBTA at 1740 cm-1 shift to 1736 cm-1 in the co-crystal. Meanwhile, the characteristic peak at 3633 cm-1 corresponding to the stretching vibration of v-OH in S-ofloxacin shifts to 3672 cm-1 in D-DBTA:S-OFLX co-crystal. Compared with the FT-IR experimental results, the calculated results offer supporting evidence to show the the bond vibration frequency for hydrogen bonding formed in co-crystal. According to the PXRD patterns, it is easy to conclude that every specific co-crystal has its unique crystal structure. The co-crystals formed in this study were 16


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characterized by PXRD. The PXRD patterns of RS-oflxoacin, S-oflxoacin, D-DBTA, L-DBTA and all the co-crystals are shown in Figure 11. It can be seen that each co-crystal formed by ofloxacin and DBTA shows a unique PXRD pattern, which is significantly different from the PXRD patterns of ofloxacin and DBTA, confirming the formation of a new co-crystal. In addition, the thermodynamic stability of co-crystals was studied by the differential scanning calorimetry (DSC) analysis. As shown in Figure 12, the melting point of L-DBTA:S-ofloxacin co-crystal (Tpeak = 140.4 °C) is higher than that of D-DBTA:S-ofloxacin co-crystal (Tpeak =138.0 °C). As shown in Figure 12d, the first endotherm peak at 137.8 °C is corresponding to the melting point of D-DBTA:S-oflxoacin co-crystal, while the second endotherm peak at 149.8 °C is supposed to be the melting point of D-DBTA:R-oflxoacin co-crystal. Similarly, the melting point of L-DBTA:R-ofloxaicn co-crystal should be 150.8 °C, as shown in Figure 12f. However, the melting points of S-ofloxacin, D-DBTA and L-DBTA are 218 °C, 155.6 °C and 155.4 °C, respectively. Therefore, the melting point of each co-crystal differs from that of API and the conformer, confirming the formation of new co-crystals, which agrees with the results of PXRD characterization. Since the hydrogen bond between ofloxacin and DBTA was formed, the lattice energy of co-crystals decreased sharply, resulting in obtaining lower melt points of the co-crystals than that of ofloxacin and DBTA. The diastereomeric pair of D-DBTA:R-ofloxacin and D-DBTA:S-ofloxacin co-crystals have not only different crystal structures, but also different properties like thermal behaviour. 17



The thermogravimetric analyses of ofloxacin, DBTA and co-crystals were performed and the results are shown in Figure 13. It can be seen that a mass loss of co-crystal could be first observed in each TGA curve. The melting point observed in DSC curve of co-crystal was accompanied by the mass loss observed in TGA curve, indicating that the co-crystal might be less stable than D-DBTA and S-ofloxacin. Recently, the mechanism of chiral discrimination has been studied by the crystal structure analysis.42 The co-crystal structure of L-DBTA:S-OFLX was determined by PXRD using Material Studio 8.0. An Rwp value of 7.53 was obtained for L-DBTA:S-OFLX (Figure 14). L-DBTA:S-OFLX crystallized in the space group P2 of the monoclinic crystal system and the unit cell consisted of two L-DBTA and two S-OFLX molecules (Figure 15a) and the crystallographic parameters are listed in Table 3. It can be seen from Figure 15b, a heterosynthon (O–H…O) between the – C=O group of L-DBTA and the -OH group of the S-OFLX could be found in the L-DBTA:S-OFLX co-crystal. Similarly, another hydrogen bonding was fomred between the –C=O group of S-OFLX and the -OH group of L-DBTA. Likewise, as shown in Figure 16 and Figure 17, an Rwp value of 7.28 was obtained for D-DBTA:S-OFLX during the structure determination. D-DBTA:S-OFLX crystallized in the space group P2 of monoclinic crystal system. The crystallographic parameters are listed in Table 4. Furthermore, the equilibrium solubility determination of co-crystals prepared by the slurry method was conducted. As shown in Figure 18, the equilibrium solubility of the formed co-crystal is significantly lower than that of S-OFLX and tartaric acid 18


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derivatives, indicating that the co-crystals could be prepared by the slurry crystallization in aqueous solution. Moreover, the equilibrium solubility of L-DBTA:S-OFLX co-crystal is lower than that of D-DBTA:S-FOLX co-crystal, which demonstrates that L-DBTA can selectively co-crystallize with the S-OFLX. Finally, the comparison of the resolution performances of the co-crystal method in this study and other enantioseparation methods reported previously for ofloxacin is listed in Table 5. Compared with those results reported in literatures, the separation method proposed in this work is efficient and clean (water as solvent), which indicates that the proposed clean method is a considerable method for resolution of racemic ofloxacin with advantages of being environmentally friendly. CONCLUSIONS In this study, a novel and green co-crystallization process for separating racemic ofloxacin in aqueous solution was proposed by using DBTA as chiral selectors. Effects of chiral selector type, resolution time, amounts of chiral selectors and racemic ofloxacin, and temperature on resolution performance were studied. D-DBTA and L-DBTA showed higher separation factor and % ee values for racemic ofloxacin than the other used tartaric acid derivatives. These two chiral selectors could form diastereomeric co-crystal pairs with two enantiomers of ofloxacin, which showed distinct physicochemical properties like the melting point and solubility. The resolution process could be carried out at a moderate temperature and reached equilibrium at 50 min. A maximum 81.8% ee for S-enantiomer in the aqueous phase was obtained with 0.08 g D-DBTA as chiral selector, while a maximum 82.3% ee for 19



R-enantiomer in the aqueous phase was obtained with 0.08 g L-DBTA as chiral selector at 278.2 K. The characterizations by FT-IR, PXRD, DSC and TGA demonstrated the formation of diastereomeric co-crystallizations between DBTA and ofloxacin in aqueous solution. In addition, the mole ratio of D-DBTA or L-DBTA to oflxoacin in co-crystals was determined as 1:1 by slurry crystallization. D-DBTA and L-DBTA selectively co-crystallized with the R-enantiomer and S-enantiomer in aqueous solution, respectively. Since significant differences in the stability and solubility in water existed between DBTA:R-OFLX and DBTA:S-OFLX, S-enantiomer and R-enantiomer could be separated efficiently in aqueous solution by co-crystallization, suggesting a resolution method with no addition of organic solvent. The resolution technology for racemic ofloxacin via forming co-crystal in the aqueous phase was more efficient and environmentally benign than previous chiral resolution methods, suggesting a novel technique that might be extended to separate other racemic compounds which can’t form salts. ASSOCIATED CONTENT Supporting Information Liquid chromatographic diagrams for the separation and detection of ofloxacin stereomers; DFT calculation results for molecular electrostatic potential (MEP); DFT calculation results of FT-IR spectra for D-DBTA, S-OFLX, and co-crystal D-DBTA:S-OFLX.

AUTHOR INFORMATION Corresponding Author *E-mail: (Zhongqi Ren) [email protected] 20


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* E-mail: (Zhiyong Zhou) [email protected]

ORCID Zhongqi Ren: 0000-0002-2571-5702 Zhiyong Zhou: 0000-0001-6436-1399 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21576010, 21606009 and U1607107), Beijing Natural Science Foundation (2172043),

Fundamental

(BUCTRC-201515)

and

Research State

Key

Funds

for

Laboratory

the of

Central

Universities

Chemical

Engineering

(SKL-ChE-16A03). The authors gratefully acknowledge these grants. REFERENCES (1) Ager, D. J. Handbook of Chiral Chemicals, Marcel Dekker, New York, 2005. (2) Lorenz, H.; Seidel-Morgenstern, A. Processes to separate enantiomers. Angew.

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27



List of Tables Table 1 Thermodynamic parameters for the separation process of S-OFLX and R-OFLX with D-DBTA and L-DBTA as chiral selectors at the temperature ranging from 278 K to 323 K Table 2 The mass balance of slurry crystallization experiment Table 3 Crystallographic parameters of L-DBTA: S-OFLX Table 4 Crystallographic parameters of D-DBTA: S-OFLX Table 5 Comparison with other separation methods related to ofloxacin enantiomers

28


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Table 1 Thermodynamic parameters for the separation process of S-OFLX and R-OFLX with D-DBTA and L-DBTA as chiral selectors at the temperature ranging from 278 K to 323 K ∆H°(KJ/mol)

∆S°(J/mol)

∆G°(KJ/mol)

R2

S-OFLX

-18.09

-41.30

(-4.74 to -6.60)

0.993

R-OFLX

-25.62

-48.40

(-9.98 to -12.16)

0.988

S-OFLX

-27.14

-54.22

(-9.62 to -12.06)

0.994

R-OFLX

-19.41

-46.30

(-4.44 to -6.53)

0.984

Parameter D-DBTA

L-DBTA

29



Page 30 of 55

Table 2 The mass balance of slurry crystallization experiment Final composition Compound

Initial

Aqueous

composition

phase (× 10

(× 10-5 mol)

mol)

Solid -5

phase(× 10-5

DBTA/OFLX (mol/mol)

mol)

D-DBTA:S-OFLX

1.01

D-DBTA

14.01

9.57

4.44

S-OFLX

6.92

2.53

4.39

D-DBTA:RS-OFLX

0.995

D-DBTA

14.99

9.22

5.77

S-OFLX

3.46

0.84

2.62

R-OFLX

3.46

0.28

3.18

L-DBTA:S-OFLX

1.06

L-DBTA

11.17

4.32

6.85

S-OFLX

6.92

0.47

6.45

L-DBTA:RS-OFLX

1.06

L-DBTA

11.17

3.21

7.96

S-OFLX

5.53

0.75

4.78

R-OFLX

5.53

2.83

2.70

30


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Table 3 Crystallographic parameters of L-DBTA: S-OFLX Parameter

L-DBTA:S-OFLX

Chemical formula Stoichiometry Formula weight Temperature (K) Crystal system Space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) Vol. (Å3) 2θ range

C18H20FN3O4·C18H14O8 1:1 719.67 Room temperature Monoclinic P2 17.9267 7.26426 14.2744 90 97.3639 90 1843.54 5o-45o

31



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Table 4 Crystallographic parameters of D-DBTA: S-OFLX Parameter

D-DBTA:S-OFLX

Chemical formula Stoichiometry Formula weight Temperature (K) Crystal system Space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) Vol. (Å3) 2θ range

C18H20FN3O4·C18H14O8 1:1 719.67 Room temperature Monoclinic P2 17.0121 8.76745 14.4693 90 107.892 90 2053.76 5o-45o

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Table 5 Comparison with other separation methods related to ofloxacin enantiomers Methods

Separation factor

% ee

Reference

1.63

-

[43]

2.48

40.4%

[44]

1.32

-

[45]

44%

[46]

82.3%

This work

(α) Ionic liquid-assisted ligand-exchange chromatography Enantioseparation by mixed extractants in biphasic system Enantioseparation in aqueous two-phase system Enantioseparation using biomacromolecules Enantioselective co-crystallization in

10.78

aqueous solution

33



List of Figures Figure 1. The hydrogen bonding in the 1 : 1 D-DBTA : S-ofloxacin co-crystal. (remarks: gray, white, red, blue and lightblue balls represent C atom, H atom, O atom, N atom and F atom, respectively.) Figure 2. Effect of chiral selector type on resolution performance. (Temperature = 298.2 K, 25 mL 1.6 g·L-1 initial oflxoacin aqueous solution, 0.08 g initial chiral selectors, resolution time = 50 min.) Figure 3. Variations in separation factor and % ee with respect to resolution time. (a, D-DBTA as chiral seletor, b, L-DBTA as chiral selector. Temperature = 298.2 K, 25 mL 1.6 g·L-1 initial oflxoacin aqueous solution, 0.08 g initial chiral selectors.) Figure 4. Variations in separation factor and % ee with respect to the amount of chiral selectors. (a, D-DBTA as chiral seletor, b, L-DBTA as chiral selector. Temperature = 298.2 K, 25 mL 1.6 g·L-1 initial oflxoacin aqueous solution, resolution time = 50 min.) Figure 5. Variations in separation factor and % ee with respect to the concentration of ofloxacin aqueous solution. (a, D-DBTA as chiral seletor, b, L-DBTA as chiral selector. Temperature = 298.2 K, 0.08 g initial chiral selectors, resolution time = 50 min.) Figure 6. Variations in separation factor and % ee with respect to temperature. (a, D-DBTA as chiral selector, b, L-DBTA as chiral selector. 0.08 g initial chiral selectors, 25 mL 1.6 g·L-1 initial ofloxacin aqueous solution, resolution time = 50 min.)

34


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Figure 7. Variations in yield in the aqueous phase with respect to temperature. (a, D-DBTA as chiral selector, b, L-DBTA as chiral selector. 0.08 g initial chiral selectors, 25 mL 1.6 g·L-1 initial ofloxacin aqueous solution, resolution time = 50 min.) Figure 8. Linear regression of ln K vs. 1/T. (a, D-DBTA as chiral selector, b, L-DBTA as chiral selector. 0.08 g initial chiral selectors, 25 mL 1.6 g·L-1 initial ofloxacin aqueous solution, resolution time = 50 min.) Figure 9. MEP mapped on the van der Waals surface for structures of uncharged molecules of S-OFLX (a) and D-DBTA (b). The unit of color gradient is hartree. Figure 10. FT-IR spectra of the co-crystals (a) D-DBTA:S-OFLX, (b) L-DBTA:S-OFLX, (c) D-DBTA:RS-OFLX, and (d) L-DBTA:RS-OFLX prepared by slurry reaction experiments. Figure 11. PXRD patterns of the co-crystals (a) D-DBTA:S-OFLX, (b) L-DBTA:S-OFLX, (c) D-DBTA:RS-OFLX, and (d) L-DBTA:RS-OFLX prepared by slurry reaction experiments. Figure 12. DSC curves of (a) D-DBTA, (b) L-DBTA, (c) D-DBTA:S-OFLX, (d) D-DBTA:RS-OFLX, (e) L-DBTA:S-OFLX, and (f) L-DBTA:RS-OFLX. Figure 13. TGA curves of the co-crystals of (a) D-DBTA:S-OFLX, (b) D-DBTA:RS-OFLX, (c) L-DBTA:S-OFLX, and (d) L-DBTA:RS-OFLX prepared by slurry reaction experiments. Figure 14. X-ray intensity for L-DBTA:S-OFLX as a function of 2θ. (The experimental pattern, the calculated (best Rietveld fit profile) pattern, reflection

35



positions and the difference curve between the experimental and the calculated patterns (Rwp value = 7.53%) are marked as blue, red, green and black, respectively.) Figure 15. (a) The arrangement of molecules in the unit cell for L-DBTA:S-OFLX. (b) The formation of H-bonding in the L-DBTA:S-OFLX co-crystal. Figure 16. X-ray intensity for D-DBTA:S-OFLX as a function of 2θ. (The experimental pattern, the calculated (best Rietveld fit profile) pattern, reflection positions and the difference curve between the experimental and the calculated patterns (Rwp value = 7.28%) are marked as blue, red, green and black, respectively.) Figure 17. (a) The arrangement of molecules in the unit cell for D-DBTA:S-OFLX. (b) The formation of H-bonding in the D-DBTA:S-OFLX co-crystal. Figure 18. Experimental solubilities of S-OFLX, RS-OFLX, L-DBTA, D-DBTA, L-DBTA:S-OFLX co-crystal and D-DBTA:S-OFLX co-crystal in aqueous solution.

36


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Figure 1. The hydrogen bonding in the 1 : 1 D-DBTA : S-ofloxacin co-crystal. (remarks: gray, white, red, blue and lightblue balls represent C atom, H atom, O atom, N atom and F atom, respectively.)

37



Figure 2. Effect of chiral selector type on resolution performance. (Temperature = 298.2 K, 25 mL 1.6 g·L-1 initial oflxoacin aqueous solution, 0.08 g initial chiral selectors, resolution time = 50 min.)

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Figure 3. Variations in separation factor and % ee with respect to resolution time. (a, D-DBTA as chiral seletor, b, L-DBTA as chiral selector. Temperature = 298.2 K, 25 mL 1.6 g·L-1 initial oflxoacin aqueous solution, 0.08 g initial chiral selectors.)

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Figure 4. Variations in separation factor and % ee with respect to the amount of chiral selectors. (a, D-DBTA as chiral seletor, b, L-DBTA as chiral selector. Temperature = 298.2 K, 25 mL 1.6 g·L-1 initial oflxoacin aqueous solution, resolution time = 50 min.)

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Figure 5. Variations in separation factor and % ee with respect to the concentration of ofloxacin aqueous solution. (a, D-DBTA as chiral seletor, b, L-DBTA as chiral selector. Temperature = 298.2 K, 0.08 g initial chiral selectors, resolution time = 50 min.)

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Figure 6. Variations in separation factor and % ee with respect to temperature. (a, D-DBTA as chiral selector, b, L-DBTA as chiral selector. 0.08 g initial chiral selectors, 25 mL 1.6 g·L-1 initial ofloxacin aqueous solution, resolution time = 50 min.)

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Figure 7. Variations in yield in the aqueous phase with respect to temperature. (a, D-DBTA as chiral selector, b, L-DBTA as chiral selector. 0.08 g initial chiral selectors, 25 mL 1.6 g·L-1 initial ofloxacin aqueous solution, resolution time = 50 min.)

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Figure 8. Linear regression of ln K vs. 1/T. (a, D-DBTA as chiral selector, b, L-DBTA as chiral selector. 0.08 g initial chiral selectors, 25 mL 1.6 g·L-1 initial ofloxacin aqueous solution, resolution time = 50 min.)

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Figure 9. MEP mapped on the van der Waals surface for structures of uncharged molecules of S-OFLX (a) and D-DBTA (b). The unit of color gradient is hartree.

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Figure 10. FT-IR spectra of the co-crystals (a) D-DBTA:S-OFLX, (b) L-DBTA:S-OFLX, (c) D-DBTA:RS-OFLX, and (d) L-DBTA:RS-OFLX prepared by slurry reaction experiments.

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Figure 11. PXRD patterns of the co-crystals (a) D-DBTA:S-OFLX, (b) L-DBTA:S-OFLX, (c) D-DBTA:RS-OFLX, and (d) L-DBTA:RS-OFLX prepared by slurry reaction experiments.

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Figure 12. DSC curves of (a) D-DBTA, (b) L-DBTA, (c) D-DBTA:S-OFLX, (d) D-DBTA:RS-OFLX, (e) L-DBTA:S-OFLX, and (f) L-DBTA:RS-OFLX.

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Figure 13. TGA curves of the co-crystals of (a) D-DBTA:S-OFLX, (b) D-DBTA:RS-OFLX, (c) L-DBTA:S-OFLX, and (d) L-DBTA:RS-OFLX prepared by slurry reaction experiments.

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Figure 14. X-ray intensity for L-DBTA:S-OFLX as a function of 2θ. (The experimental pattern, the calculated (best Rietveld fit profile) pattern, reflection positions and the difference curve between the experimental and the calculated patterns (Rwp value = 7.53%) are marked as blue, red, green and black, respectively.)

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Figure 15. (a) The arrangement of molecules in the unit cell for L-DBTA:S-OFLX. (b) The formation of H-bonding in the L-DBTA:S-OFLX co-crystal.

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Figure 16. X-ray intensity for D-DBTA:S-OFLX as a function of 2θ. (The experimental pattern, the calculated (best Rietveld fit profile) pattern, reflection positions and the difference curve between the experimental and the calculated patterns (Rwp value = 7.28%) are marked as blue, red, green and black, respectively.)

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Figure 17. (a) The arrangement of molecules in the unit cell for D-DBTA:S-OFLX. (b) The formation of H-bonding in the D-DBTA:S-OFLX co-crystal.

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Figure 18. Experimental solubilities of S-OFLX, RS-OFLX, L-DBTA, D-DBTA, L-DBTA:S-OFLX co-crystal and D-DBTA:S-OFLX co-crystal in aqueous solution.

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Table of Contents Graphic Manuscript title: "Green and Efficient Resolution of Racemic Ofloxacin using Tartaric Acid Derivatives via Forming Co-crystal in Aqueous Solution" Lichao He, Zhongrui Liang, Guojia Yu, Xiangrong Li, Xinjian Chen, Zhiyong Zhou* and Zhongqi Ren*

Manuscript ID: cg-2018-00414v.

A novel and green co-crystallization process for separating racemic ofloxacin by using tartaric acid derivatives as chiral selectors in aqueous solution was proposed. The separation technology for ofloxacin via forming diastereomeric co-crystals was more efficient and environmentally benign than previous chiral resolution methods, suggesting a novel technique that might be extended to separate other racemic compounds which can’t form salts.

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Green and Efficient Resolution of Racemic Ofloxacin using Tartaric

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