Conformational Flexibility and Crystallization: The Case of Furosemide

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Conformational Flexibility and Crystallization: The Case of Furosemide Na Tang, Xuemin Wang, Wei Du, Lei Zhang, Jun Xiang, Songbo Wang, Penggao Cheng, Liang Zhu, and Qiuxiang Yin Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01407 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on March 3, 2019

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Crystal Growth & Design

Conformational Flexibility and Crystallization: The Case of Furosemide Na Tang†, Xuemin Wang†, Wei Du†,*, Lei Zhang†, Jun Xiang†, Songbo Wang†,Penggao Cheng†, Liang Zhu†, Qiuxiang Yin ‡

† College of Chemical Engineering and Materials Science, Tianjin Key Laboratory of Marine Resources and Chemistry, Tianjin University of Science & Technology ， Tianjin 300457, People's Republic of China, and ‡School of Chemical Engineering and Technology, Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Tianjin University, Tianjin 300072, People's Republic of China *Email: [email protected] Abstract The nucleation and growth processes of Furosemide crystals in crash cooling crystallization, evaporative crystallization and anti-solvent crystallization were investigated to evaluate the effects of molecular conformational flexibility on the crystallization process and the polymorphic formation. Powder X-ray diffraction, Thermogravimetric analysis, solid ATR-FTIR, and Scanning Electron Microscope were used to characterize three Furosemide polymorphs, the one of which was obtained in pure form only once. The relationship between the three forms was determined from both the thermogravimetric analysis data and the solution-mediated polymorphic transformation experiments. It was found that both forms II and III are monotropically related with Form I, and Form I is the most stable form at the investigated temperature range. Crystallization of Furosemide was studied in 10 organic solvents at different temperatures, supersaturatons and pressures to gain a general view of the polymorphic outcome and the ease of the crystallization. The energy barrier to go from conformer II or III to conformer I in solution is estimated to be about 15kJ∙mol-1, which is moderate at ambient conditions for the transition,

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while that to go from conformer II to conformer III is calculated to be around 2kJ∙mol-1, which indicates the interconversion between II and III facile. It was found that in solution crystallizations pure Furosemide Form I and Form II can be obtained, while Form III cannot be obtained in pure forms and arises concomitantly or together with Form I, and even the three forms arise together. The molecular conformational flexibility is not a problem for the polymorphic crystallization of Furosemide. Finally, the pathway to produce different polymorphs of Furosemide

was paved to outline the differences of different conformational polymorphs. Keywords:

conformational

flexibility;

polymorphism;

nucleation;

growth;

conformation transition 1. Introduction The increasing interest in developing pharmaceutically active compounds having molecular weights greater than 300 has led to significant activity in exploring the relationship between conformational flexibility and crystallization mechanisms. There is a strong sense, for example that molecules having significant conformational freedom may be difficult to crystallize. The relationship between conformational flexibility and crystallization mechanism has aroused wide interests due to the importance of molecular conformation in the chemistry of the organic solid state and the strong link between conformation in the crystal structure and solution.1-8 Because conformational flexible molecules have more degrees of freedom than rigid molecules, multiple conformers of a flexible molecule can exist in equilibrium with each other in solution and a greater scope for polymorphism might be expected.

9-10

According to the classical nucleation theory, the nucleation process can be envisioned as a multistep process in which molecules first associate into clusters whose structures resemble that of the final crystals and then assemble into crystal nuclei which finally grow into crystals.11-12 Thus, it is an undoubted question in terms of crystallization that is conformational flexibility a problem for crystallization? Yu et al.

13

argued that the crystal structure plays a key role in determining which


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conformer in solution is expected to crystallize and considered that multiple conformers in solution is likely to hinder the crystallization process. This can be explained by the fact that a number of different conformations may be present in solution, which makes the crystallization process more complicated since the process of crystallization must select the “right” conformers among the “wrong” ones. When different conformers result to different crystal structures, the effective concentration of the conformer which crystallizes into the observed polymorph is lowered, leading to the decrease of the supersaturation ratio of that conformer.

6-8

Threlfall et al.14

observed the comparative crystallization behaviour of more than 400 related acylanilides to gain a view of the importance of conformation in the crystallization time for organic compounds. They speculated that the number of alternative orientations in which a molecule can dock at a crystal growing site dominates the ease of crystallization. If one conformer can rotate itself to dock onto another conformer’s growing site, the retardation of crystal growth will be expected less. In solution, the population of conformers depends on conformational energies, energy barriers of transition between conformers, temperature and solvent-solute interactions.7 When multiple molecular conformations can be stabilized in crystalline, conformational polymorphism arises.13, 15 Conformational polymorphism is defined as the existence of different conformers of the same molecule in different polymorphic modification, wherein polymorphs differ not only in the mode of packing but also on molecular conformation.1,

9, 16

To properly address the subject of conformational

polymorphism, one must address the definition of the terms “conformation” and “conformer”. A conformation is the spatial arrangement of the atoms affording distinction between stereoisomers which can be interconverted by rotations about formally single bonds, whereas a conformer is one of a set of stereoisomers, each of which is characterized by a conformation corresponding to a distinct potential energy minimum.

17

When referring to conformations in crystal structures, it is important to

differentiate between conformational adjustment and conformational change. The former always occurs in a crystal for any flexible molecule which adjusts to the



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crystal environment by slightly varying its conformation to minimize the lattice energy of the crystal with a small conformational energy penalty while the latter involves a change of gas-phase conformer:

It requires going uphill on the gas-PES;

over the energy barriers; downhill into a different potential energy well; and finally, conformational adjustment of the new gas-phase conformer to its crystal structure.

18

Thus, to probe the relationship between conformational flexibility and crystallization mechanism, it should be firstly understood that when conformational change might determine the ease of nucleation and crystal growth and secondly the related process of conformational polymorphism should be explained. The chemical name of the model compound in this work, Furosemide, is 4-chloro-2-[(2-furanylmethyl)-amino]-5-sulfamoylbenzoic acid. It is a diuretic widely used in the treatment of congestive heart failure and edema，and several structurally characterized polymorphs have been reported in the literatures and Cambridge Structural Database (reference no.FURSEM01-03, FURSEM12-18). The molecular structure of Furosemide (Figure 1) gives an indication of the molecule’s ability to exhibit conformations. In furosemide, sulfonamide group and furan ring torsions can result in different conformers in solution, increasing the probability of conformational polymorphism in solid state. There are mainly three polymorphs (I, II, III), two solvates (DMF solvate and 1,4-dioxane solvate) and one amorphous form.16,19 The polymorphic behavior and the crystallization process of Furosemide has barely been reported.


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Figure 1. The chemical diagram of furosemide, the red and yellow molecules correspond to the two conformers of FURSEM03, the green molecule and the blue molecule represent the conformers in FURSEM14 and FURSEM16 respectively. In this study, the thermodynamic relationship between form I and the ideal solubility of Furosemide was firstly identified. Then crash cooling crystallization, evaporative crystallization and anti-solvent crystallization in different solvents at different temperatures and supersaturations were investigated to gain a general view of the polymorphic outcome and the ease of the crystallization of Furosemide, the solution of which contains multiple conformers. Finally, the energy barrier of conformational change between different conformations was discussed and how a molecule of one conformation might dock onto a surface of the other one’s structure to see if conformational change is possible for a docked molecule. 2. Experimental Section 2.1 Materials and Process Analysis Tools. Furosemide Form I (CAS no.54319, >98% purity) was purchased from Shanghai Macklin Biochemical Co., Ltd and used without further purification. Furosemide Form II was obtained by standing the n-butanol solution (dissolve 2.00g Furosemide Form I in 100 g of n-butanol at 338.15K) at 283.15K. Furosemide Form III was obtained by crash cooling the 2-propanol solution (S=5) from 338.15K to 278.15K and maintained for 24h. The 1,4-dioxane solvate was crystallized by crash cooling the 1,4-dioxane solution. The



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Furosemide forms were polymorphically pure as judged by their powder X-ray diffraction (PXRD) patterns. All of the solvents were purchased from Tianjin Jiangtian Chemical Technology Co. Ltd., China, and of analytical reagent grade and the molar purities were > 99.5%. PXRD was performed using a SHIMADZU Lab X-6100 X-ray powder diffractometer at a wavelength of 1.5406 Å controlled by jade 6 software from 5° to 40° at a scan speed

of

2.000°∙min-1.

Differential

Scanning

Calorimetry

(DSC)

and

thermogravimetric analysis (TGA) experiments were performed using either a Mettler Toledo DSC 30 instrument controlled by Mettler TC15 complete with a liquid nitrogen cooling system with data analyzed by STARe software v.610 or a TA DSC Q100 with software universal analysis 2000 v. 4.5A. Infrared spectra were performed using a BURKER TENSOR27 fourier transform infrared spectrometer and controlled by OPUS software. Scanning Electron Microscope (SEM) experiments were prepared by a KYKY SBC-12 Sputter Coater and were conducted by the Phenom Pure Desktop SEM. Hot-stage microscope (HSM) experiments were conducted by a GMR-213P SHGMYQ polarizing microscope and the temperature is controlled by a KER3100-08S precision constant temperature table. 2.2 Solubility Measurements. The solubility of Furosemide Form I in 8 organic solvents was measured as functions of temperature in the range of 278.15-333.15 K. Excess amounts of pure Form I were added to 20mL of solvent to saturate the solutions and create a slurry. The desired temperature was maintained by a thermostatic water bath (XOTS-2006, Nanjing Xianou Instruments Manufacture Co., Ltd, China) with an accuracy of ±0.1 K. After being stirred for 4 h at each temperature, the suspension was allowed to settle for 30 min. Then a 5-mL sample of the clear solution was taken using a syringe with a membrane filter (0.20 µm). The residue of undissolved crystals was separated and identified to be the initial polymorph

by

PXRD,

indicating

that

no

solvent-mediated

polymorphic

transformation occurred during solubility measurement experiments. Samples of the saturated solution were dried at room temperature until the solvent was completely


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evaporated. The solubility was determined from the mass of the remaining crystalline material and the total solution. 2.3 Crash Cooling Crystallization. The crystallization of Furosemide was investigated in crash cooling crystallizations in 1, 4-dioxane, methanol, ethanol, 2-propanol and n-butanol. These experiments were carried out using a 50 mL jacketed vessel with an overhead 2-blade impeller stirring at 200 rpm. Solutions at different concentrations were prepared by dissolving the corresponding amount of Furosemide Form I in 40g solvent. The solutions were kept at about 333.15 K for 1 h to ensure that all the crystals were dissolved completely. 20 mL aliquots of the solutions were then withdraw and filtered through a pre-heated 0.2 µm syringe filter, transferred to the jacketed vessel pre-set to the desired crystallization temperature (XOTS-2006, Nanjing Xianou Instruments Manufacture Co., Ltd, China). The crystals were filtered immediately after the crystals accumulated enough to measure PXRD and dried at room temperature for 0.5 h. The residual slurry was maintained at the desired temperature for several hours to check the solution-mediated polymorphic transformation. Each experiment was repeated 5 times and PXRD was applied to identify the polymorphic forms of the product crystals. 2.4 Evaporative Crystallization. The evaporative crystallization of Furosemide was investigated in acetone, 1,4-dioxane, methanol, ethanol, 2-propanol, n-butanol, acetonitrile, acetic acid and ethyl acetate at different pressures. 100g saturated solution at room temperature was transferred into a 500mL rotary evaporator(IKA RV 10 digital) at a rotary speed of 60 rpm. The crystals were separated immediately after nucleation and examined by PXRD to determine the crystal form. 2.5 Anti-solvent Crystallization. The crystallization of Furosemide was investigated in anti-solvent crystallization with distilled water as the anti-solvent and methanol, ethanol, 2-propanol, acetone, 1, 4-dioxane, DMF and acetonitrile as the solvent, respectively. The desired amount of Furosemide was dissolved into the solvent and kept at desired temperature, and then a certain amount of water was added. The precipitated crystals were collected by filtration and identified via PXRD.



2.6 Solvent-mediated Polymorphic Transformation. The solvent-mediated polymorphic transformation experiments from Form II to Form I of Furosemide were investigated on 100 mL jacketed vessel with an overhead 2-blade impeller stirring at 200 rpm. The effects of solvent on crystal transformation was examined among methanol, ethanol, 2-propanol and 1,4-dioxane at 303.15 K by measuring the transformation time, which is defined as the time from the addition of Form II to the finish of transformation. 20 mL slurry was sampled at intervals, filtered immediately and the crystals to measure PXRD. During each experiment, 100 mL of different solvent was initially saturated with respect to form I. An extra 5.0 g of Form II was then added into the saturated solution. 3. Results and Discussion 3.1 Identification of Furosemide Polymorphs. The PXRD patterns of the unground pure white crystals obtained in the experiments are proved to be pure Form I, Form II and Form III compared with the calculated ones (FURSEM03, FURSEM16 and FURSEM14, respectively) in Mercury, which is shown in Figure 2. This confirms them to be pure Forms I, II and III respectively. It is noted that some intense peaks in the calculated patterns are essentially missing from the experimental patterns. This is certainly due to preferred orientation since the crystals of both forms are known to be needles.


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Figure 2. Powder X-ray diffraction patterns of unground Furosemide crystals compared to the calculated patterns: (a) Form I and FURSEM03, (b) Form II and FURSEM16 and (c) Form III and FURSEM14. Figure 3 shows the infrared spectra of the Furosemide polymorphs. The absorption band in the region 3200-3400 cm-1 was characterized fully for crystal forms; it can be



observed correspondence with the results where the secondary amine N-H stretching vibration remains unaltered between Forms I, Form II and Form III, the absorption band attributable to the vibration of 3350 cm-1. The apparent lack of a sulphonamide N-H vibration absorption band near 3400 cm-1 is a characteristic for form III and the another sulphonamide N-H vibration absorption peak moved to a lower wave number 3254 cm-1, compare with 3285 cm-1 for form I. The change in the vibrations associated with the sulphonamide group suggests that there is an alteration in the hydrogen bond sequence within the crystal. This may result from a change in molecular conformation and crystal packing.20 1,4-dioxane solvate exhibited absorption peak at 1125 cm-1 ascribed to the C-O-C of 1,4-dioxane, indicative of their presence as solvate.

Figure 3.The FTIR spectra of Furosemide polymorphs. The SEM images of furosemide crystals are given in Figure 4. Distinct morphology differences were evident among these samples. Form I presents two crystal morphologies, platelike (major) and needles to block (a few), and both From II and Form III crystals are needle-like, which makes it impossible to identify the polymorphs just via morphology determination.


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Figure 4. SEM of furosemide pure Form I, mixed crystals of Form I and Form II, mixed crystals of Form I and Form III, and pure Form III. The magnifications are 1000×. 3.2 The Thermodynamic Relationship of Forms I, II and III. The weak endothermic peak in the DSC of Form I at 131.9oC results from a solid-solid phase transition from Form I to a stable form at higher temperature19 (Figure 5). This behavior was also found in the DSC of concomitant polymorphs of Form II, Form III at 127.2oC and 188.6oC, respectively. The DSC curve of 1,4-dioxane solvate shows two strong endothermic peaks at 95.6oC and 108.7oC, a weak endothermic peak at 139.8oC. A 19.2% loss in total weight indicated by TG curve suggests that the endothermic peaks are caused by desolvation and the value is very close to that of 21.03% determined theoretically for a 1:1 dioxane solvate. The PXRD of a desolvated sample is identical with form I. The weak endothermic peak at 139.8oC was therefore attributable that of the resultant form I undergoing transformation to a stable form at higher temperature. According to the heat of transition rule21, the existence of a solid-solid phase transition below the melting point suggests the polymorphs have an enantiotropic relationship. Above the observed solid-solid transition temperatures, the stable form at higher temperature stated



melting and then decomposition at 212.2-223.7oC. The 1,4-dioxane solvate is difficult to preserve for a long time. From the solution-mediated transformation experiments in 2-propanol it was found that both Form II and Form III transformed into Form I within 24h at examined temperatures. Taken together these data indicate that Form I, Form II and Form III are all enantiotropically related with the stable form at higher temperature. Form II and Form III are monotropically related with Form I, and Form I is the stable form among the three forms at temperature range of 0 to 80oC.

Figure 5. DSC and TG curves of furosemide polymorphs: (a) DSC, (b) TG. To further verify the solid - solid phase transition around 132°C, a hot-stage microscope was applied to visualize the heating process of Furosemide polymorphs shown in Figure 6, which demonstrates the typical solid - solid phase transition. It can


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be seen from Figure 6 that the morphology of Form II transformed into dark state from the original transparent crystals, indicating the crystal structure modification during heating and Form II transformed into a more stable form at higher temperatures.

Figure 6. HSM snapshots of furosemide form II crystals 3.3 Solubility. Equilibrium solubility data of Furosemide Form I in methanol, ethanol, 2-propanol, ethyl acetate, 1,4-dioxane, n-butanol, acetonitrile and acetic acid were determined by gravimetric method.22-23 The experimental solubility data are listed in Supporting Information Tables S1 and graphically plotted in Figure 7. Van’t Hoff equation was applied to fit the experiment data and the relative contribution of enthalpy and entropy to the standard Gibbs energy during the dissolution process were calculated. It can be clearly seen from Figure 7 that the solubility of Furosemide in all the solvents increases with the increasing of temperature. The solubility in 1,4-dioxane is the highest and that in acetic acid is the lowest among the 8 organic solvents. The solubility of Form II and Form III are not available due to the stability, which makes it impossible to evaluate the Gibbs free energy between the polymorphs. As shown in Table S2, Table S3, ∆disH, ∆dis S, ∆disG are positive in all cases. It can be inferred from ∆disH﹥0 that the dissolving process in endothermic, which is in good agreement with the phenomenon that solubility of Furosemide Form I increases as the temperature increases. It is obvious that ∆disH makes greater contributions to ∆disG



than ∆disS (%ζH﹥59.74 and up to 83.63). Thus, the dissolving process of Furosemide Form I is enthalpy-driven.

Figure 7. Mole fraction solubility of furosemide in eight pure solvents:□, 1,4-dioxane; ○, methanol;△, ethanol; ▷,ethyl acetate;▽, 2-propanol; ☆, acetonitrile; ◇n-butanol; ◁, acetic acid; solid lines, correlated data by the modified Apelblat equation. 3.4 Solvent-mediated Polymorphic Transformation. According to Ostwald’s rule, the metastable form is likely to nucleate first during the crystallization process. It will then transform into the more stable form over time. Thus, it is necessary to determine whether the transformation between form I and form II of and form II to 1,4-dioxane solvate of Furosemide will happen and whether the formation of concomitant polymorphs is due to the transformation process. The results of solvent-mediated polymorphic transformation in different solvents at 30oC are shown in Figure 8. The induction times during transformation from Form II to Form I in methanol, ethanol and 2-propanol were 15min, 2h and 21h, respectively. It took 2h in methanol and 17h in ethanol for the completely transformation from Form II to Form I and it took only 1h in 1,4-dioxane for that of Form II to the solvate.


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Figure 8. PXRD patterns of samples with time. The samples were collected at different time during solution-mediated transformation experiment of Furosemide Form II at 30oC in different solvents. 3.5 Crystallization Outcomes. The polymorphic outcomes of crash cooling crystallization, evaporative crystallization and anti-solvent crystallization at different temperatures and supersaturations are compared here with full and detailed results given in Figure 9, Table S4, Table 1 and Table 2. Note that the temperature scale of Figure 9 is not intended to be linear. Although it was hoped to cover the same supersaturation range for each solvent and each temperature this proved to be impossible as a result of kinetic limitations. Thus, for example, at low temperatures crystallization did not occur within 24 hours at low supersaturations while at high temperatures and high supersaturations crystallization had begun before the set temperature was reach. It is noted that typically the induction times for precipitation


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are minutes compared to transformation times between forms which are of the order of hours. This means that the forms arise from direct precipitation not via transformation. 3.5.1 Crash Cooling Crystallization. The polymorph outcomes of crash cooling crystallization in methanol, ethanol, 2-propanol, n-butanol and 1,4-dioxane at different temperatures (T) and supersaturations (S) were studied. Detailed results are given in the Table S4 whilst the crystallization results are presented for four sets of temperatures (5oC, 15oC, 25oC and 40oC) in Figure 9. It can be seen from Table S4 and Figure 9 that pure Furosemide Form I can always be obtained in methanol and ethanol at the tested temperatures (T), pure Form II can be obtained in n-butanol, while Form III cannot be obtained in pure form and the form arise concomitantly or together with Form I, and even the three forms arise together. Form I and Form II were obtained together at the supersaturation range of 2.2-3.2 in methanol and 2.5-6 in ethanol. Considering the results of solution-mediated transformation and the sampling time, the formation of the mixture of Form I and Form II is rationally assumed to arise concomitant nucleation. In n-butanol, only Form II was obtained. In 1,4-dioxane, only solvate was obtained. At the investigated supersaturation with the sampling time range from minutes to hours, indicating a strong link between polymorph and solvent. In 2-propanol, from I and form III arise together at relatively high supersaturation and the amount of crystals was not enough for PXRD testing at low supersaturation. Note that increasing the sampling time results in bigger amount of form I in the mixtures and the pure form I can be obtained when sampling time was up to 10 h for the mixture of form I and form II and up to 48 h for the mixture of form I and form III, indicating that form I is the stable form at the investigated temperatures and the transformations from Form II/III to From I are not easy. The formation of Furosemide polymorphs in crash cooling crystallization seems to obey the Ostwald’s rules that the stable form (Form I) inclines to precipitate at low supersaturation and the metastable form (Form II and Form III) tends to precipitate at high supersaturation, which is not quite sure due to the lack of process analysis tools



to identify the trace crystals at the moment of nucleation.

Figure 9. Crash cooling crystallization outcomes as a function of solvent, supersaturation and temperature. S” represents the supersaturation ratio corresponding to Form I. The squares correspond to experiments for which at least 3 out of the five crystallizations resulted in the same form. 3.5.2 Evaporative Crystallization. The polymorph outcome of evaporative crystallization of Furosemide in 9 organic solvents at different temperatures (T) and pressures (P) were investigated. The induction times were not recorded in detail and they are of the order of 20 minutes. Detailed results are given in Table 1, from which it can be seen that pure Form I can always be obtained on the investigated crystallization conditions in all solvents except 2-propanol, n-butanol and ethyl acetate, and Form II arises always concomitantly with Form I. It seems that the


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concomitant polymorphism of Form I and Form II was apt to precipitate at lower evaporative speed while pure Form I tended to nucleate at higher evaporative speed, which obeys to the Ostwald’s rules. The solvent has no effect on the polymorphic outcome and Form III was not obtained in all the experiments. Table 1.The polymorph outcome of evaporative crystallization (■represents pure Form I, ▲represents concomitant polymorphism of Form I and II and ●represents concomitant polymorphism of Form I and 1,4-dioxane solvate) Solvent

Temperature (℃)

Pressure (MPa)

acetone

25 30

1,4-dioxane

65

-0.065 0.1 -0.078 -0.081

0.1

25 methanol

-0.065 -0.075 -0.076 -0.080 -0.081 -0.084 -0.086 -0.090 -0.071 -0.076 -0.081 -0.082 -0.070 -0.072

45

ethanol

50

2-propanol

52

n-butanol

80

acetonitrile

50

acetic acid

80

ethyl acetate

50

Polymorph Outcome ■ ■ ● ● ▲ ▲ ■ ▲ ■ ▲ ▲ ▲ ▲ ■ ■ ■ ■ ▲ ▲

3.5.3 Anti-solvent Crystallization. The anti-solvent, distilled water, was added into the saturated solutions immediately at room temperature and the molar fraction ratios of solvent to water are from 1:1 to 1:24 to reach a high supersaturation at which Furosemide precipitated instantly.

The results of polymorph outcome are given in

Table 2, from which it can be seen clearly that pure Form I was obtained in 1,4-dioxane, acetonitrile, acetone and DMF. Form I and Form II arise concomitantly



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in methanol and ethanol while Form I and Form III form concomitant polymorphism in 2-propanol, which is similar to the polymorph outcome in crash cooling crystallization. Table 2. The polymorph outcome of anti-solvent crystallization (■ represents pure Form I, ▲represents concomitant polymorphism of form I and II and ● represents concomitant polymorphism of Form I and III) Solvent 1,4-dioxane methanol ethanol 2-propanol acetonitrile acetone DMF

Molar ratio (solvent to anti-solvent) 1:18 1:1 1:4 1:6 1:12 1:3 1:24

Induction time instantly instantly instantly instantly instantly instantly instantly

Polymorph Outcome ■ ▲ ▲ ●

■ ■ ■

3.5.4 Difficulty Level of Crystallization. The induction times measured in crash cooling crystallization were observed to be among seconds to hours and dependent on solvent, supersaturation and temperature. The results are shown in Figure 10. Obviously, increasing supersaturation results in shorter induction times at the given solvent and temperature (Figure 10a), and increasing temperature generates shorter induction times at the given solvent and supersaturation (Figure 10b). At high temperatures and high supersaturations Furosemide nucleated in less than a minute such as the nucleation in methanol when T=15oC and S=3.0 while it took more than 3 days for Furosemide to nucleate at low temperatures and low supersaturations, such as the nucleation in ethanol when T=5oC and S=2. Although the conformational flexibility of Furosemide may cause a problem for its crystallization, nucleation of Furosemide polymorphs seems not affected. It obeys the classical nucleation theory that ln(tind) has a linear relationship with 1/(ln2S), which can be regressed from the data in Table S4. The shorter induction times lead to higher nucleation speeds and arise in solvents in which the solubility of Furosemide is higher. The growth of Furosemide seems affected strongly considering the long lag of time to get enough


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amounts of crystals to examine PXRD in spite of the fact that polymorphic transformation occurred with time.

Figure 10.The profile of induction time with supersaturation a) in five solvents at 288.15K and b) in ethanol at 3 sets of temperatures during crash cooling crystallization In all experiments of the crash cooling crystallization, evaporative crystallization and anti-solvent crystallization, Form I was favored to precipitate or to be obtained due to the stability. Form II can be obtained in n-butanol by cooling crystallization in n-butanol. Form III was barely obtained. Form II and Form III arise together with Form I on the most condition from concomitant nucleation. Considering the lattice energies (-41.65, -41.78 and -41.53 kcal∙mol-1 for Form I, Form II and Form III,



respectively) calculated by Babu et al16, the very little energy differences are readily to result in concomitant polymorphism, which can explain the experiment results rationally. Conformers Transition Barrier. To probe how much the conformational flexibility of Furosemide molecules may affect the crystallization process, the transitions between different conformers are taken into consideration. The energy barrier to go from conformer II or III to conformer I in gas phase is estimated from Babu’s data16 to be over than 20 kJ·mol-1, which is very high and so unlikely to occur at ambient conditions for the transition24, while that to go from conformer II to conformer III is calculated to be around 5 kJ·mol-1, which indicates that the interconversion between II and III facile (Figure12). To determine the conformer transition barriers in solvent environment, conformer energies were calculated in Gaussian 09 (B3LYP/6-31G*) using implicit solvent model25. Furosemide is truncated to its N-methyl derivative at the N-CH2-furyl portion. The energy of conformer III is arbitrarily set to 0, the energy profile with the torsion angles between SO2NH2 group and N-methylchloroanthranilic acid is plotted in Figure 12. The conformation energy calculated for the molecule is listed in Table 3. Calculating the percentage of different Furosemide conformations according to Boltzmann distribution, the result is listed in Table 4. The energy barriers to go from conformer II or III to conformer I in different solvents are estimated from Figure 12 to be about 15 kJ∙mol-1, which is moderate at ambient conditions for the transition, while that to go from conformer II to conformer III is calculated to be around 2 kJ∙mol-1, which indicates the interconversion between II and III facile (Figure 12). From this point of view, it is rational to speculate that in the investigated solvents the Furosemide conformers I, II and III may transform into each other without costing too much energy, which makes the conformational flexibility of Furosemide not a problem for its crystallization. In addition, the molecular structure of tolfenamic acid24 is similar to furosemide, the crash cooling crystallization results of which are shown in Supporting Information Figure S1. The nucleation results are similar with that of Furosemide, which leads to draw the conclusion that the


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conformational flexibility of Furosemide in solution does not affect the overall polymorphic outcome. In this case the combination of solvent, temperature and supersaturation influences the formation of conformational polymorphs of Furosemide, which results in a negligible effect of the presence of multiple conformations in solution on crystallization behavior. Thus, the pathway to produce different polymorphs of Furosemide was paved to outline the differences of different conformational polymorphs, which can be illustrated in Figure 13.

Figure 11. The interconversions between different conformers of Furosemide

Figure 12. Furosemide is truncated to its N-methyl derivative at the N-CH2-furyl portion. Conformer energies are plotted for every 10° change in sulfonamide torsion between 0 and 360° Table 3. Conformer torsion anglea (from X-ray structure), computed conformer energyb (Gaussian 09, B3LYP/6-31G*), the lowest energy conformer is arbitrarily set



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to 0 Conformer

Conformer energy/kJ·mol-1

Torsion angle

water

1,4-dioxane

methanol

ethanol

2-propanol

n-butanol

acetone

form I

166.1

5.858

8.197

6.239

6.307

6.302

6.376

6.706

form II

79.9

1.902

2.767

1.742

1.987

2.113

2.065

2.096

form III

55.7

0

0

0

0

0

0

0

Table 4.Percentage of different conformations Conformer form I form II form III

Percent (%) water 5.632 29.378 64.990

1,4-dioxane methanol 2.423 4.749 23.375 31.033 74.203 64.218

ethanol 4.766 28.928 66.306

2-propanol n-butanol 4.847 4.680 27.852 28.297 67.301 67.023

acetone 4.116 28.208 67.675

Figure 13.The pathway to produce Furosemide polymorphs 4. Conclusions The effects of molecular conformational flexibility on the crystallization process and the polymorphic formation were studied in this work from the aspects of crash cooling crystallization, evaporative crystallization and anti-solvent crystallization. It was found that in solution crystallizations pure Furosemide Form I can always be obtained, and Form II can be obtained in n-butanol at higher supersaturations and arose together with Form I on the most condition from concomitant nucleation, while Form III was barely obtained in pure forms and the form arise concomitantly or together with Form I, and even the three forms arise together. The energy barriers of conformer transitions in solvent environment among conformers I, II and III were


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calculated, which indicates that the transitions among the three conformers are facile in solution. Therefore, it is rational to speculate that the effect of conformational flexibility of Furosemide on polymorph crystallization behavior is negligible.

■ AUTHOR INFORMATION Corresponding Author *Tel.: 86-22-60602731. Fax: 86-22-60602731. E-mail: [email protected] . ■ ACKNOWLEDGEMENTS The authors are grateful for the financial support of the National Natural Science Foundation of China (No.21506162), Tianjin Municipal Natural Science Foundation (No.17JCQNJC13200) and Key Project of Tianjin Municipal Natural Science Foundation (No.16JCZDJC32700). ■ SUPPORTING INFORMATION Solubility of forms I are listed in Table S1, Table S2 and Table S3, respectively. This information is available free of charge via the Internet at http://pubs.acs.org/.



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For Table of Contents Use Only Conformational Flexibility and Crystallization: The Case of Furosemide Na Tang, Xuemin Wang, Wei Du, Lei Zhang, Jun Xiang, Songbo Wang, Penggao Cheng, Liang Zhu, Qiuxiang Yin

Synopsis: The effects of molecular conformational flexibility on the crystallization and the polymorphic formation of Furosemide were taken into account as well as supersaturation, temperature and solvent. The crystallization thermodynamics, kinetics and polymorphic transformation were studied. The relationship between different polymorphs and the pathway to produce different polymorphs of Furosemide was paved to outline the differences of different conformational polymorphs.


Conformational Flexibility and Crystallization: The Case of Furosemide

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