Laboratory Experiment pubs.acs.org/jchemeduc
High-Resolution Solid-State NMR Spectroscopy: Characterization of Polymorphism in Cimetidine, a Pharmaceutical Compound Julia E. Pacilio, John T. Tokarski, Rosalynn Quiñones, and Robbie J. Iuliucci* Department of Chemistry, Washington and Jefferson College, Washington, Pennsylvania 15301, United States S Supporting Information *
ABSTRACT: High-resolution solid-state NMR (SSNMR) spectroscopy has many advantages as a tool to characterize solid-phase material that finds applications in polymer chemistry, nanotechnology, materials science, biomolecular structure determination, and others, including the pharmaceutical industry. The technology associated with achieving high resolution has evolved to where SSNMR spectroscopy has become routine. To highlight SSNMR spectroscopy capability, an experiment exploring polymorphism in a pharmaceutical compound is described. Polymorphism can be studied by one-dimensional 13C NMR spectroscopy, presenting a straightforward experiment to highlight the techniques of cross-polarization, magic-angle spinning, and decoupling. To aid those unfamiliar with solid-state NMR methods, a detailed tutorial on the associated techniques is provided. The polymorphs of cimetidine, the active pharmaceutical agent of Tagamet, were selected to study. The development of the histamine H2-receptor antagonist was novel, and the rational drug design approach led Sir James W. Black to share the 1988 Nobel Prize in Physiology and Medicine. Because some of the polymorphic forms fail to produce crystals suitable for X-ray diffraction, SSNMR spectroscopy has played a critical role in characterizing the crystal structures of cimetidine polymorphs. The experiment has been implemented for an advanced analytical laboratory. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Spectroscopy, NMR spectroscopy, Solids, Drugs/Pharmaceuticals
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polymorphism to the pharmaceutical industry and its reliance on SSNMR spectroscopy make an interesting application relevant to biochemists and prehealth students, in addition to chemists preparing for industrial careers.
olecular structure is the key to chemical properties, the reactivity of compounds, and the function of biological systems. Because nuclear magnetic resonance (NMR) spectroscopy serves as one of the leading techniques to elucidate molecular structure, the coverage of NMR spectroscopy is a requirement in the undergraduate curriculum. It is not surprising to find the bevy of experiments and articles that describe NMR spectroscopy in this Journal. Structure plays just as important a role in solid-phase materials, and the need to characterize functional solids finds applications in polymer chemistry, nanotechnology, and materials science, as well as structural biology. With the basic concepts of NMR spectroscopy being taught in the first two-years of a standard college curriculum, students are more prepared to learn advanced NMR techniques in upper-level laboratory exercises. Only three laboratory experiments have been reported in this Journal that focus on solid-state NMR (SSNMR)1 spectroscopy and none of these experiments emphasize the cross-polarization pulse sequence used to achieve high resolution. As the commercial technology associated with SSNMR spectroscopy continues to evolve, the presence of capabilities at educational communities will continue to increase. The goal here is to expose undergraduates to cross-polarization under magic-angle spinning. Because the characterization of polymorphs can be achieved with 1D 13C SSNMR spectroscopy, polymorphism is ideal to demonstrate the technique. The importance of © XXXX American Chemical Society and Division of Chemical Education, Inc.
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BACKGROUND: DRUG AND NMR The feasibility to characterize drug tablets has promoted SSNMR spectroscopy to be one of the leading tools to analyze polymorphic forms of pharmaceutical compounds.2 Because solid structure affects a drug’s manufacturing process, shelf life, and appropriate administration, the proper identification of the structure is important for drug development. Insufficient knowledge of polymorphism in ritonavir, an antiretroviral drug, resulted in a drug transformation to the thermodynamically more stable, but ineffective, polymorph. 3 Abbot Laboratories experienced a “market crisis”discovering manufacturing plants contaminated and the need to recall its product worldwide. The ritonavir incident changed the pharmaceutical industry forever. Cimetidine, which is the active ingredient in Tagamet, also has many polymorphic forms that potentially affect its commercial production. Sir Black’s “rational drug design” applied to Tagamet set the precedent for future pharmaceutical
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research.4 The histamine H2 receptor antagonist provides relief to patients with gastroesophageal reflux disease. The structure of cimetidine (Figure 1) contains a flexible guanidine chain and numerous hydrogen bonding sites to
Figure 1. Stick figure of cimetidine shows the conformation of polymorphic Form A with the position numbering. The color scheme of white, black, blue, and yellow is used to represent hydrogen, carbon, nitrogen, and sulfur, respectively.
Figure 2. Impact of the CP/MAS experiment on a sample of 3methylglutaric acid is shown. The spectrum on the left was acquired without CP/MAS conditions and was signal-averaged overnight. The CP/MAS spectrum on the right required only 30 s. The pulse sequence is shown in the center.
promote the formation of different crystalline structures that depends on solvent polarity and recrystallization conditions. To date, seven polymorphic forms of cimetidine have been identified.5 Approximately 20% of all organic compounds fail to crystallize in ways suitable for single-crystal diffraction studies. Cimetidine is one such problematic compound; only three crystal structures of the four anhydrous forms and one of the three monohydrate hydrates have been resolved by diffraction. NMR crystallography attempts to address this issue.6 Previous SSNMR and molecular modeling studies were used to resolve the polymorphic structures of cimetidine.7 Because of its inherent resolution, liquid-phase has dominated the applications of NMR spectroscopy for decades. The stochastic motion of molecules in a liquid is fast on the time scale of an NMR experimentaveraging the line broadening. For spin 1/2 nuclei, dipolar coupling and chemical shift interactions are present. These are anisotropic properties; their magnitude depends on the orientation of the molecule with respect to the field. In a powder, the molecules remain fixed and are randomly aligned. Each site becomes described by an envelope of frequencies; line widths can be ten to hundreds of kilohertz wide. The peaks can be wider if quadrupolar nuclei are being observed. Broad peaks dramatically decrease the resolution, preventing useful interpretation of the spectrum. The signal-to-noise is reduced, further degrading the spectral quality. To achieve high-resolution, both mechanical motion of the sample and irradiation with high-power radio frequency (RF) must be employed. The spectral enhancement these techniques provide is demonstrated in Figure 2. The act of spinning a solid at an angle of 54.74° with respect to the field “magically” eliminates those anisotropic interactions. To remove them completely, the spinning rate must exceed the size of the interaction, or spinning sidebands appear. Resolution is further enhanced by RF irradiation to eliminate strong heteronuclear dipolar couplings. Narrow peaks in SSNMR spectroscopy can still exceed one ppm and further signal enhancement is desired. The signal of naturally low abundant isotopes, e.g., 13C nuclei, can be increased by cross-polarization where magnetization from the abundant proton spin reservoir is transferred to the
rare spin. High-resolution SSNMR spectroscopy of rare spin-1/ 2 nuclei typically refers to the combination of cross-polarization under magic-angle-spinning (CP/MAS) conditions and highpower decoupling. Although plenty of SSNMR explanations exist, a guide to setting up CP/MAS conditions is hard to find in the literature. For this reason, a tutorial in the Supporting Information (SI) is provided.
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EXPERIMENT Cimetidine was commercially obtained. The polymorphic forms of cimetidine were prepared on the basis of the procedures of Bauer-Brandle.5 Form A is recrystallized from acetonitrile. Water is used to prepare Forms B, C, and M1. The rate of cooling, initial temperature, and concentration are critical factors to obtain the desired form. A 15% (w/w) aqueous solution heated to 70 °C and left to cool crystallizes as Form B. Both Forms C and M1 recrystallize from a 5% (w/w) aqueous solution. If warmed (70 °C) and rapidly cooled (liquid nitrogen bath), Form C is obtained. Otherwise, the monohydrate form (M1) is recovered. All spectra were acquired using a 400 MHz NMR spectrometer. A 4 mm double resonance MAS probe was simultaneously tuned to 1H (399.952 MHz) and to 13C (100.568 MHz) for the cross-polarization pulse sequence where the 90° pulses were set to 2.5 μs. A 3 ms contact period was used under 10 kHz of magic-angle-spinning. 3-Methylglutaric acid (MGA) was used to set up the experiment.8 Cimetidine utilized a 13.25 s repetition rate, and MGA used 3 s. The 13C spectra are referenced externally to TMS by measuring the resonance frequency of the methyl carbon in MGA.8
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HAZARDS One must be aware of all hazards associated with high magnetic fields and high-power RF when performing NMR spectroscopy. Details on these hazards are outlined in the SI. The usual laboratory safety protocols should be utilized, given the use of organic solvents (acetonitrile). B
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RESULTS AND DISCUSSION The experiment was conducted successfully in an upperdivision undergraduate analytical laboratory course two times. Ten undergraduate students completed the experiment. Because of the need to share instruments, it is routine to schedule the tasks of an experiment over multiple periods of an analytical course. For this experiment, sample preparation was conducted during down-time of prior periods, one lab period was designated to acquire the NMR spectra, and the NMR processing was done collectively in a computer lab during a third course period. Students were assigned one polymorph (one assigned to commercial Tagamet), which they prepared, characterized by melting point, and IR spectroscopy. The preparation and characterization steps required less than an hour. To accommodate more students, small groups (2 or 3) could perform the experiment collectively. Undergraduates with “good” laboratory skills were able to obtain high yields of the four forms (A, B, C, and M1). However, all student attempts to prepare Form D resulted in Form B. Each individual/group explored SSNMR conditions of magic-angle spinning, crosspolarization, and decoupling by acquiring 13C spectra of MGA (Figure 3). The spectra were processed by students and
Figure 4. 13C Solid-State NMR spectra of four polymorphic forms of cimetidine using the cross-polarization pulse sequence under magicangle spinning. If known, the conformation of the polymorph is shown. Suitable spectra are attainable in 15 minutes. However, spectra shown here have 3 h of signal averaging. The same color scheme identified in Figure 1 is employed here with the addition of red for the oxygen atom.
Table 1. 13C Chemical Shifts of Solid-State Forms of Cimetidine Peaks in the SSNMR spectra of the polymorphic forms (ppm) assignment
A
B
C
M1
C4 C5 C9 C6 C7 C3 C2 C10 C1 C8
10.6 23.3 27.9 28.4 41.2 119.2 120.8 135.0 135.9 161.1
9.1, 14.0 20.6, 27.7 26.7, 28.4 31.4, 36.3 40.7 119.3, 120.1 124.7, 128.2 132.2, 133.7 134.8 160.8
8.5, 9.6 27.4, 28.1 31.1, 31.6 35.8 41 120, 127.5 128.4 133.6, 134.4 132.4 160.6
10.0 26.8 28.6 28.6 41.6 121.8 126.2 131.4 135.1 159.1
cross-polarization became evident in the 13C spectrum with no cross-polarization. Polymorphism was studied in the context of hydrogen bonding in cimetidine that alters the chain conformation and the tautomerism associated with the imidazole ring. Because the microcrystalline samples of cimetidine had a cotton-like morphology, the feasibility of single-crystal diffraction was discussed in contrast to high-resolution SSNMR spectroscopy. Drug manufacturing is an appropriate topic to cover with Tagamet. Students identified the polymorph form in Tagamet as Form A (Figure 5). Drug stability during manufacturing is an issue as polymorphs are susceptible to mechanical stress and this subject has been studied in cimetidine.5
Figure 3. Conditions of high-resolution SSNMR spectroscopy were explored with MGA by decreasing the MAS by 1/3 (green), turning off cross-polarization (aqua), and lowering the decoupling by 1/2 (purple). Each spectrum was signal-averaged for only five min for a total of 20 min.
analyzed for resolution and signal-to-noise. Each group collected one spectrum of their polymorph or commercial sample (Figure 4). The set of 5 spectra (4 MGA and one cimetidine) required 35−45 min of NMR time. Polymorphs were identified by comparing chemical shifts of their spectra to those in Table 1. Both the chemical shift anisotropy and the H−C dipolar couplings are significantly large in most organic compounds that their presence would make it impossible to extract any useful information from a solid-phase 13C NMR spectrum. With instructor supervision, students investigated three main features of high-resolution SSNMR spectroscopy by acquiring four 13C spectra of MGA. The degradation of resolution and signal-to-noise were examined by reducing the proton decoupling power. The position and appearance of spinning sidebands were discussed in association with the spectrum where the MAS was lowered. Signal-to-noise enhancement of
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CONCLUSION With the increasing frequency of SSNMR spectroscopy appearing in the literature, it is relevant to expose undergraduates to the most common experimentCP/MAS conditions. The topic of polymorphism demonstrated the value of CP/MAS conditions without resorting to multidimensional spectroscopy. By discussing polymorphism, industrial challenges, such as those observed by pharmaceutical companies, were emphasized. A pharmaceutical example was selected deliberately to attract interest from biochemists. C
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REFERENCES
(1) (a) Anderson, S. E.; Saiki, D.; Eckert, H.; Meise-Gresch, K. A solid-state NMR experiment: analysis of local structural environments in phosphate glasses. J. Chem. Educ. 2004, 81, 1034−1037. (b) Kinnun, J. J.; Leftin, A.; Brown, M. F. Solid-State NMR Spectroscopy for the Physical Chemistry Laboratory. J. Chem. Educ. 2013, 90, 123−128. (c) Stark, R. E.; Gaede, H. C. NMR of a phospholipid: Modules for advanced laboratory courses. J. Chem. Educ. 2001, 78, 1248−1250. (2) (a) Wenslow, R. M.; Baum, M. W.; Ball, R. G.; McCauley, J. A.; Varsolona, R. J. A spectroscopic and crystallographic study of polymorphism in an aza-steroid. J. Pharm. Sci. 2000, 89, 1271−1285. (b) Harris, R. K. Applications of solid-state NMR to pharmaceutical polymorphism and related matters. J. Pharm. Pharmacol. 2007, 59, 225−239. (3) (a) Bauer, J.; Spanton, S.; Henry, R.; Quick, J.; Dziki, W.; Porter, W.; Morris, J. Ritonavir: an extraordinary example of conformational polymorphism. Pharm. Res. 2001, 18, 859−866. (b) Chemburkar, S. R.; Bauer, J.; Deming, K.; Spiwek, H.; Patel, K.; Morris, J.; Henry, R.; Spanton, S.; Dziki, W.; Porter, W.; Quick, J.; Bauer, P.; Donaubauer, J.; Narayanan, B. A.; Soldani, M.; Riley, D.; McFarland, K. Dealing with the impact of ritonavir polymorphs on the late stages of bulk drug process development. Org. Process Res. Dev. 2000, 4, 413−417. (4) Page, C. P.; Schaffhausen, J.; Shankley, N. P. The scientific legacy of Sir James W. Black. Trends Pharmacol. Sci. 2011, 32, 181−182. (5) Bauer-Brandl, A. Polymorphic transitions of cimetidine during manufacture of solid dosage forms. Int. J. Pharm. 1996, 140, 195−206. (6) Harris, R. K., Wasylishen, R. E., Duer, M. J., Eds. NMR Crystallography; John Wiley & Sons Ltd.: Chichester, 2009; p 504. (7) Middleton, D. A.; Le Duff, C. S.; Peng, X.; Reid, D. G.; Saunders, D. Molecular conformations of the polymorphic forms of cimetidine from 13C solid-state NMR distance and angle measurements. J. Am. Chem. Soc. 2000, 122, 1161−1170. (8) Barich, D. H.; Gorman, E. M.; Zell, M. T.; Munson, E. J. 3Methylglutaric acid as a 13C solid-state NMR standard. Solid State Nucl. Magn. Reson. 2006, 30, 125−129.
Figure 5. 13C Solid-State NMR spectra of cimetidine Form A (red) and Tagamet (green) superimposed. With the exception of the extra peaks in 60 to 105 ppm range due to the inactive ingredients found in the tablet, the spectra are identical.
Students made the general connection for the growing industrial need to analyze materials and the value of SSNMR spectroscopy to do so. Success of this experiment was gauged by a student’s ability to articulate the learning objectives (e.g., the techniques of high-resolution SSNMR spectroscopy, structural verification based on chemical shifts, conditions leading to polymorphism, and the importance of polymorphism to the pharmaceutical industry). This experiment was designed for instrumental analysis, but modifications could see its incorporation into physical chemistry or biochemistry. It is peculiar to find the technique of cross-polarization absent from the repertoire of experiments in this Journal. This absence may stem from the challenges of acquiring a CP/MAS spectrum. Therefore, a detailed tutorial on the set up of the NMR spectrometer for a CP/MAS experiment is provided in the SI.
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ASSOCIATED CONTENT
S Supporting Information *
Instructor and student’s notes include information for a multiweek advanced laboratory experiment. A tutorial on how to set up a CP/MAS experiment is provided. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: riuliucci@washjeff.edu Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the RUI−NSF Chemistry Program (NSF Grant CHE-0956755). We are grateful for additional financial support from the College President and administration. We would like to thank Arno Kentgen and his research group at Radboud University, The Netherlands, for access to a high magnetic field spectrometer. International travel to The Netherlands was provided by NSF’s Office of International Science and Engineering. We would like to acknowledge Duquesne University’s Center for Research and Education in X-ray Diffraction (NSF Grant No. DUE0511444) for access to their diffraction facilities. D
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