Sweet Theophylline Cocrystal with Two Tautomers ... - ACS Publications

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Sweet Theophylline Cocrystal with Two Tautomers of Acesulfame Lin Wang,†,‡ Min Luo,§ Jianhui Li,† Jianming Wang,§ Hailu Zhang,*,† and Zongwu Deng† †

Laboratory of Magnetic Resonance Spectroscopy and Imaging, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P.R. China ‡ College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P.R. China § Crystal Pharmatech, Suzhou Industrial Park, Suzhou 215123, P.R. China S Supporting Information *

ABSTRACT: A novel pharmaceutical cocrystal (THPAH12) of Theophylline (THP) was obtained with an artificial sweetener, Acesulfame (AH), in a molar ratio of 1:2. Solid state NMR spectra of the cocrystal indicate that the two AH molecules exist as keto and enol tautomers, which is further confirmed by the refined crystal structure. THPAH12 is the first keto form AH containing cocrystal. This highlights the fact that not only −OH of enol form of AH, but also the −NH− CO group of the keto form of AH should be considered when designing new pharmaceutical cocrystals via the supramolecular synthon approach. Compared with pure THP, THPAH12 possesses enhanced solubility and hydration stability, which highlight its potential for further pharmaceutical applications.

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in its keto form (AH keto, Scheme 1b) in both of the two solid forms,19 while for the pharmaceutical cocrystal of AH, all three reports (five cocrystals of AH) show that the AH molecule is in its enol form (AH enol, Scheme 1c).15,17 Thus, it is of interest to find a cocrystal in which AH exists in the keto form. The supramolecular synthon approach20 and structural resemblance strategy21,22 are often employed for cocrystal design and screening. A 1:1 pharmaceutical cocrystal of Theophylline (THP, Scheme 1a) with Saccharin (SAC) was reported recently, in which an R22(9) synthon formed between C2O2, N4H of THP, and −NH−CO group of SAC (Scheme 1e).23 Such an R22(9) graph-set motif (or its analogues) has been recognized as a main heterosynthon in cocrystals of THP.23−29 Similar to SAC, the −N5H−C8O6 group also exists in the keto form of AH (Scheme 1b), which drives us to find out whether a cocrystal can be formed between THP and keto form of AH. The pursuance of THP/AH crystalline complex was first conducted by ethanol-assisted grinding with a starting THP/ AH ratio of 1:1. THP form II and AH form I were used in this study. With respect to the starting materials, new resonance peaks appear on the solid state 13C CP/MAS (crosspolarization/magic angle spinning) TOSS (total sideband suppression) NMR spectrum of the product (THPAH11G, Figure 1c), indicating the formation of a new phase, while this spectrum also indicates that excess THP exists in the ground

n active pharmaceutical ingredient (API) may exist in various solid forms, such as polymorphs and amorphous forms of pure API compounds, hydrates, solvates, salts, and cocrystals. As different forms have unique physicochemical properties, it is an essential step to screen and select adequate solid form for drug development. Most recently, pharmaceutical cocrystals have received attention in the pharmaceutical industry for their potential to fine-tune the physicochemical and biological properties of free API.1−4 Cocrystals are stoichiometric multicomponent systems built upon noncovalent interactions where all the components present are solid under ambient conditions.3 One topic in the cocrystal research field receiving recent attention is molecular tautomerism, though only a few examples have been reported.5−11 Both stable and metastable tautomers may be obtained by forming intermolecular interaction with surrounding molecules.5−11 Obviously, such systems contain valuable information about supramolecular interactions, which is in turn helpful to design new pharmaceutical cocrystals and to target other useful tautomeric systems.12,13 The tautomeric molecule under consideration in the current report is Acesulfame (AH). Acesulfame is an aliphatic caloriefree sweetener and its potassium salt is widely used in food products, beverages, and pharmaceutical formulations. The sweetness of acesulfame is approximately 200 times greater than that of sucrose.14 AH has been used as a guest molecule to prepare novel pharmaceutical crystalline complexes.15−18 In addition to the pleasant sweet taste,15 these complexes were also reported possessing enhanced solubility and physical stability.16−18 AH was found to exist in two polymorphic forms, and X-ray structural analysis revealed that the AH molecule is © XXXX American Chemical Society

Received: February 10, 2015 Revised: May 14, 2015

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DOI: 10.1021/acs.cgd.5b00207 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Scheme 1. Molecular Structures of THP (a), AH (b−d), and a Proposed R22(9) Synthon between THP and AH (e)

enol form, while further investigation is needed in order to confirm the state of the other AH molecule. 15 N CP/MAS NMR spectra provide more valuable information. With the formation of THPAH12, the chemical shift of one N5 atom of AH changes from ∼ −230 ppm (−227.3 and −229.6 ppm, AH keto, Figure 2b) to −179.2 ppm

Figure 1. 13C CP/MAS TOSS NMR spectra of THP (Form II) (a), AH (Form I) (b), THPAH11G (c), and THPAH12 (d).

product (indicated by dash lines). Therefore, the starting ratio of THP/AH was adjusted to 1:2, and a pure new crystal sample (THPAH12, Figure 1d) was obtained. The formation of pure crystalline THPAH12 was also confirmed by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) measurements (SI Figures S1 and S2). There are other important clues in the 13C spectrum of THPAH12. Each chemically distinct carbon in THPAH12 is represented by single resonance, indicating the number of molecules per asymmetric unit (Z′) of the complexes should be equal to 1. Chemical shifts for C8 and C8′ (also for C9/C9′, C10/C10′) are different, indicating that the two THP molecules are in different configurations. Remarkably, the chemical shift of one C9 atom changes from 102.2 ppm (C9, AH keto) to 94.1 ppm (C9′, THPAH12). Generally, the repacking of molecules in different crystal cells often leads to a smaller 13C chemical shift variation (less than 5 ppm). Such a larger change (−8.1 ppm) must originate from the change of tautomeric (AH enol) or ionization (AH−) state of one AH molecule. For the AH− anion, two AH salts show their C9″ chemical shifts at ∼102 ppm.16 Thus, we may preliminarily deduce that one AH molecule in THPAH12 should be in the

Figure 2. 15N CP/MAS NMR spectra of THP (Form II) (a), AH (Form I) (b), and THPAH12 (c).

(N5′, Figure 2c), and the other N5 atom keeps its resonance signal almost unchanged (−228.7 ppm, N5, Figure 2c). For the AH− anion (Scheme 1d), the 15N chemical shift of N5″ atom should appear at ∼ −200 ppm.16 Thus, the two AH molecules in THPAH12 should be in the keto form and enol form, respectively. Since there is no intermolecular proton transfer occurring, the THPAH12 complex can be classified as a cocrystal. In the THP crystal (Figure 3a), N3 is involved in a hydrogen bond with N4−H of another THP molecule. Since the 15N chemical shift for N3 atom in THPAH12 is shifted slightly upfield (Figure 2c), it is reasonable to assume that this N site is involved in a stronger hydrogen bonding interaction due to the shielding effect on the nucleus, or a modified hydrogenbonding structure.30 The 15N chemical shifts of N4 for THP B

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CD3OD, the changes are negligible. It is assumed that the interactions between AH and CD3OD reduce the probability of AH−THP interactions in the CD3OD solvent. For CDCl3, the interaction sites with AH are absent. Then, chemical shift changes caused by AH−THP interactions may be detected in CDCl3 solvent. The most important information can be obtained from these spectra is that AH is always in one form in CDCl3 or CD3OD, regardless of whether THP is present or not. The results indicate that molecular tautomeric configuration and intermolecular interactions between API and conformer may be different when they are in solution and in a crystalline state, due to the presence of compound−solvent interactions and the absence of lattice effects in solution. Finally, the crystal structure of THPAH12 was obtained via XRPD refinement (Rwp = 4.16%, Rp = 2.98%; SI Figure S5 and Table S1). The refined structure reveals that THPAH12 crystallizes in the triclinic space group (P1̅). Each asymmetric unit (Figure 3c) contains one THP molecule, one AH keto molecule, and one AH enol molecule. The observed intermolecular interactions (Figure 3c) agree with the spectral evidence. Once the crystal structure was obtained, the theoretical chemical shifts were computed. The agreement between experimental and computed 13C and 15N NMR chemical shift values (SI Table S2), especially the ones of C9′, N3, and N5′, validate the refined structure as an accurate representation of the actual cocrystal structure. For all the previously reported pharmaceutical cocrystals of AH, the AH molecule is in its enol form without exception.15,17 It is believed that the strong intermolecular hydrogen bonding between the −OH of AH enol and other molecule (−OH···O or/and −OH···N)15,17 stabilizes the enol tautomer in the solid state.17,37 Since THPAH12 contains both the enol form and the keto form of AH, it is necessary to probe the reason for the coexistence of the two tautomeric forms. DFT calculations (Gaussian09, B3LYP/6-311G(d,p) level) on molecular models were performed. The stabilizing energies (ΔE = ETHP‑AH − ETHP − EAH) of O6′H···N3 and R22(9) synthon are −52.90 and −72.00 kJ mol−1, respectively. Such an energy magnitude represents the formation of strong intermolecular hydrogen bonding. Though AH keto may have larger stabilizing energy when it interacts with other molecules, such a tautomeric form of AH (AH keto) is observed for the first time in the crystalline complexes of AH. For the five reported AH enol cocrystal systems,15,17 no suitable sites exist in those APIs, which can form geometrically matched synthons with AH keto (e.g., the R22 (9) heterosynthon). Thus, the calculated results and reported structures indicate that geometric matching, i.e., suitable supramolecular synthon, should be the main factor for the existence of AH enol or/and AH keto. The preliminary conclusion also reminds us that the supramolecular synthon strategy should be valuable guidance toward the screening of AH keto containing cocrystals. With the formation of the novel pharmaceutical cocrystal of THP, one may expect some modification of the physicochemical properties. For THP, two shortcomings are often considered. First, the solubility is not high, and some efforts are intended to improve the solubility of THP.38 For example, one of the commercially available drug substances, aminophylline, is a salt form of THP synthesized with ethylenediamine.39 Second, THP has poor physical stability against hydration and tends to form monohydrate when exposed to high relative humidity (RH) condition.40,41 These two properties are investigated here.

Figure 3. Intermolecular hydrogen bonds in THP (Form II)36 (a), AH (Form I)19 (b), and THPAH12 (c).

and THPAH12 are −218.1 and −218.7 ppm, respectively, and the 15N chemical shifts of N5 for AH keto and THPAH12 are −229.6 and −228.7 ppm, respectively. Such small changes indicate that N4−H and N5−H may still remain in hydrogen bonding interactions. According to the information for the proton donors/ acceptors in THP and AH in the previous paragraphs, one may propose that a THP molecule should interact with one AH keto molecule via the R22(9) hydrogen bonding pair (Scheme 1e), and with one AH enol molecule via N3···HO6′ hydrogen bonding in the THPAH12 cocrystal. Generally, a tautomeric molecule in a crystalline form is present exclusively in one configuration; few examples demonstrate more than one tautomeric form coexisting in one crystal structure.9,10,31−35 We should point out that the most notable aspect of this case is the existence of AH keto in the cocrystal. The result confirms that both the −OH of AH enol and the −NH−CO group of AH keto should be considered and can be used when designing new pharmaceutical cocrystals via the supramolecular synthon approach. To further confirm the structure assumption, solution cocrystallization was conducted to prepare the single crystal sample of THPAH12. Solvent evaporation (acetonitrile, methanol, ethanol, acetone, ethyl acetate, and toluene) of stoichiometric solutions was employed. However, no satisfactory results were obtained. Tautomerism is often observed in solution, and it is well-known that equilibrium between different tautomeric forms may be manipulated by changing the solvent. Thus, the solution crystallization experiments were also expected to answer whether there is a solvent effect on the cocrystal formation. It was disappointing but understandable that such crystallization experiments always yielded mixed phases of cocrystal and starting materials, because the THP and AH have significantly different solubilities in commonly used organic solvents.20 In some cases, different solid forms of AH (methanol, acetone) and THP (ethyl acetate) were detected in these crystallized products, which is interesting but outside the scope of the current study. To investigate the solvent effect on the cocrystal formation, liquid-assisted grinding with different solvents (acetonitrile, methanol, acetone, ethyl acetate, toluene, cyclohexane) was conducted, and THPAH12 was consistently obtained. It seems that there is little/no solvent effect on the outcome of crystallization. Since the cocrystal contains both AH tautomers, we expected to find out whether AH keto and AH enol also coexist in solution or not. To obtain this information, liquid 13C spectra (SI Figures S3 and S4) were collected in two commonly used deuterated solvents, CDCl3 (weak polar solvent) and CD3OD (strong polar solvent). Chemical shifts of AH change slightly when THP coexists in CDCl3. In C

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crystalline complexes of AH with simplex keto form, which is currently underway.

Figure 4 shows the comparison of the intrinsic dissolution rates (IDRs) of THP and THPAH12, which are 1.07 and 3.83

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ASSOCIATED CONTENT

S Supporting Information *

Experimental and computational procedures, XRPD patterns and DSC curves of THP, AH, and THPAH12, 13C NMR spectra of AH and THPAH12, theoretical chemical shifts and refined crystal structure of THPAH12. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b00207.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-512-62872713. Fax: +86-512-62603079. Notes

The authors declare no competing financial interest.

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Figure 4. Intrinsic dissolution profiles of THP and THPAH12 in ultrapure water at 37 °C. −2

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (No. 21205129), Jiangsu Provincial Fund for Natural Sciences (No. BK2012191), and Large Scientific Instrument Platform of Jiangsu Province (No. BZ201402). The authors also thank Platform for Characterization & Test, SINANO, CAS, and Supercomputing Center, CNIC, CAS, for the calculation supporting.

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mg cm min , respectively. The maximum apparent solubility in ultrapure water is also increased from 16.9 ± 0.3 mg·mL−1 to 73.5 ± 1.1 mg mL−1. Figure 5 shows the XRPD patterns of

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Figure 5. XRPD patterns of THP (a, c) and THPAH12 (d, e) before (a, d) and after (c, e) equilibration at 95% RH/25 °C. Simulated XRPD pattern of THP·H2O36 (b) is also given for comparison.

THP and THPAH12 after storage at high RH (95% RH/25 °C). After 24 h, anhydrous THP is completely transformed into a monohydrate, while THPAH12 keeps its original XRPD feature after equilibrating at 95% RH for one month, indicating its superior phase stability against high RH. In summary, a novel 1:2 pharmaceutical cocrystal of THP has been synthesized with AH by solvent-assisted grinding method. The spectral and structure information confirm two AH tautomers coexisting in the cocrystal sample, which is the first report of the existence of keto form AH in the crystalline complexes of AH. Improved IDR (3.6 times), solubility (4.3 times), and hydration stability of THPAH12 have been demonstrated, which highlight its potential for further pharmaceutical application. The results also confirm the effectiveness of the supramolecular strategy for control over the existence of a target tautomer, and −NH−CO group of AH can be used to design new pharmaceutical cocrystals. One effort following this contribution is to look for pharmaceutical D

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DOI: 10.1021/acs.cgd.5b00207 Cryst. Growth Des. XXXX, XXX, XXX−XXX