Characterization of Tenofovir Disoproxil Fumarate and Its Behavior

Article pubs.acs.org/crystal

Characterization of Tenofovir Disoproxil Fumarate and Its Behavior under Heating Elionai C. de L. Gomes,*,†,§ Wagner N. Mussel,† Jarbas M. Resende,† Silvia L. Fialho,‡ Jamile Barbosa,‡ Elisa Carignani,§ Marco Geppi,§ and Maria I. Yoshida*,† †

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG, 31270-901, Brazil ‡ Fundaçaõ Ezequiel Dias, Rua Conde Pereira Carneiro, 80, Belo Horizonte, MG, 30510-010, Brazil § Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Moruzzi, 3, Pisa, PI, 56124, Italy S Supporting Information *

ABSTRACT: Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor used worldwide to treat AIDS. The aim of this study was to investigate the structural and dynamic properties of the polymorphic form I of the drug by thermal analysis, Fourier transform infrared spectroscopy, solid-state NMR, and powder X-ray diffraction. A full assignment of 13C NMR resonances was achieved for the first time. Variable-temperature studies were conducted, and the results were related to changes in the TDF structure and dynamics of specific molecular fragments, especially of the fumarate portion of the molecule. It was found that, under heating, the 13C NMR signals related to the carbon atoms of the disoproxil fumarate moiety split into two resonances. The region of the phosphonate vicinity also changes. This can reflect the lack of symmetry of the two molecules in the asymmetric unit after heating. This solid−solid transformation was related to DSC and XRD observations.

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INTRODUCTION Acquired immune deficiency syndrome (AIDS) is a degenerative disease of the immune system caused by the human immunodeficiency virus (HIV), a lenti-virus belonging to the family of the Retroviridae.1,2 Tenofovir disoproxil fumarate (Figure 1) is among the most worldwide used antiretrovirals

Drug Administration (US FDA) in 2001. This drug is in clinical use for HIV infected and hepatitis B-positive patients.3,4 TDF was included in the 15th of World Health Organization (WHO) Model List of Essential Medicines5 and generally is used together with other drugs as part of the first- and secondline ARV regimen.6,7 Currently, these drugs are administrated in separate tablets; however, the WHO Committee highly recommends the use of fixed-dose combinations of ARVs (e.g., two or more active pharmacological products in the same capsule, tablet, or solution),5 but such a system could present serious interactions or incompatibilities between their components, which may be extensively investigated before the development of a stable formulation containing two or more drugs. Before the development of such a system, the full understanding of the characteristics of drugs in the solid state is necessary, so that interactions or reactions with other components, which can affect the stability, safety, and efficacy of a pharmaceutical formulation, can be predicted and avoided. The aim of this study was to investigate, at a molecular level, the structural and dynamic properties of TDF, by means of different techniques that allowed a complete characterization of the polymorphic form I of TDF in the solid state, which is used in the Viread reference medicine of TDF.

Figure 1. Chemical structure of tenofovir disoproxil fumarate.

(ARVs) for AIDS treatment. Tenofovir, C9H14N5O4P ({[(2R)1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid is a nucleoside reverse transcriptase inhibitor, while tenofovir disoproxil fumarate (TDF), C23H34N5O14P 9-[(R)2-[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate (1:1) is a prodrug form of tenofovir, which was approved by the United States Food and © 2015 American Chemical Society

Received: January 20, 2015 Published: March 3, 2015 1915

DOI: 10.1021/acs.cgd.5b00089 Cryst. Growth Des. 2015, 15, 1915−1922

Crystal Growth & Design

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Figure 2. TG and DTG curves of tenofovir disoproxil fumarate at a heating rate of 10 °C min−1 under a dynamic atmosphere of nitrogen.

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sequence). In this experiment, 400 transient for 84 rows were acquired. 31 P spectra were obtained by direct excitation with a pulse delay of 120 s and a MAS frequency of 17 kHz. All the variable-temperature experiments were acquired after 15 min of temperature stabilization. XRD. Powder X-ray diffraction (XRD) data were collected in an XRD-7000 diffractometer (Shimadzu, Japan) under 40 kV, 30 mA, using Cu Kα (λ = 1.54056 Å) equipped with a Polycapillary focusing optics under parallel geometry coupled with a graphite monochromator, scanned over an angular range of 4−70° (2θ) with a step size of 0.01° (2θ) and a time constant of 5 s step−1. The sample holder was submitted to a spinning of 30 cycles per minute to minimize rugosity effects and to reduce any eventual preferred orientation. The lattice parameters were determined by Rietveld fitting analysis, which refined 1709 reflections peaks for TDF in the range of 4−70° 2θ.

EXPERIMENTAL SECTION

Materials. Tenofovir disoproxil fumarate (TDF, C19H30N5O10P, pharmaceutical grade) was obtained from Nortec (Brazil). Methods. DSC and TG/DTG. A DSC-60 differential scanning calorimeter (Shimadzu, Japan) was used to investigate the thermal behavior of the drug. The DSC cell was calibrated with indium (mp 156.6 °C; ΔHfus = 28.54 J g−1) and lead (mp 327.5 °C). Aluminum pans containing about 1 mg of sample were used under a dynamic nitrogen atmosphere (50 mL min−1) and a heating rate of 10 °C min−1 in the temperature range from 25 to 450 °C. For the variabletemperature study, heating rates of 2, 10, and 20 °C/min were used. TG/DTG curves were obtained in a thermobalance model DTG 60 (Shimadzu, Japan) in the temperature range from 25 to 700 °C under a dynamic nitrogen atmosphere (50 mL min−1) and a heating rate of 10 °C min−1. Alumina pans with about 3 mg of sample were used. TOA (Thermo-Optical Analysis). A hot stage FP82 (Mettler, Switzerland) was used to obtain TOA images at different time intervals during heating, with a DM 4000B microscope (Leica, Germany) coupled to a Leica digital camera model DFC 280. A heating rate of 2 °C min−1 was employed, and the images were captured at 100× magnification. FTIR Spectroscopy. FTIR analysis of TDF was performed at room temperature on a Spectrum 1000 spectrophotometer (PerkinElmer, United States) equipped with an ATR (attenuated total reflectance) accessory. The sample was pressed into a zinc selenide crystal, and 32 scans were recorded from 400 to 4000 cm−1 with a spectral resolution of 4 cm−1. ssNMR Spectroscopy. All the solid-state NMR spectra were acquired on a Varian Infinity Plus 400 spectrometer operating at a Larmor frequency of 400.03, 100.59, and 161.93 MHz for 1H, 13C, and 31 P, respectively. A commercial double-resonance 3.2 mm MAS probe was used, with a 90° pulse duration of 2 μs for 1H and 31P, and of 3 μs for 13C. 1 H MAS spectra at 17 kHz were acquired by direct excitation, using a pulse delay of 5 s. Proton-decoupled 13C spectra were acquired with a ramped crosspolarization (CP) pulse sequence8 and the SPINAL decoupling scheme.9 A contact time of 5 ms was found to give the maximum signal intensity, and a pulse delay of 20 s was used in all the CP experiments. Spinning sideband-free 13C spectra were recorded using a spinning speed of 17 kHz. Non-quaternary suppression (NQS) experiments were performed using dephasing times of 50, 100, and 200 μs. A 2D MAS-J-HMQC10 spectrum was recorded with a contact time of 1 ms and a MAS frequency of 17.85 kHz (rotor synchronized

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RESULTS AND DISCUSSION Characterization of Tenofovir Disoproxil Fumarate. TG/DTG and DSC. The TG curve of TDF shows thermal stability until 138 °C. The TG/DTG curves (Figure 2) indicate that the thermal decomposition process of TDF occurs in three stages, in accordance with the following temperature ranges and weight losses: 138.1−208.1 °C (Δm = 33.95%), 208.1−273.9 °C (Δm = 12.12%), and 273.9−377.6 °C (Δm = 9.97%). After that, there is a weight loss of 9.07% due to carbonization process of the drug. The DSC curve of TDF (Figure 3) shows a sharp endothermic peak that corresponds to drug melting (Tonset = 113.5 °C; ΔHfus = 125.7 J g−1). According to the literature, the drug has at least three known polymorphic forms: forms A, B, and I.11 The melting temperature presented by the TDF sample is in accordance with the polymorphic form I, even if it is worth noticing that the melting peak reported in ref 11 is broader than that reported in Figure 3. The DSC curve of TDF shows a broad endothermic peak at 107 °C before melting (ΔHfus = 9.3 J g−1), whose assignment is not trivial: this can be attributed to (i) melting of small particles that are imperfect crystallites in the sample (common phenomenon depending on route synthesis) or to (ii) a solid−solid phase transition involving the whole sample. This issue will be better investigated in the following by variable1916



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Figure 3. DSC curve of tenofovir disoproxil fumarate at a heating rate of 10 °C min−1 under a dynamic atmosphere of nitrogen.

Figure 5. FTIR spectrum of tenofovir disoproxil fumarate.

temperature XRPD and ssNMR experiments. This broad endothermic peak at 107 °C was not observed in the previously reported DSC curve,11 but, given the width of the melting peak reported in the literature, it could be overlapped with the melting peak. The second endothermic peak at 113.5 °C should be due to in the case of (i) the melting of crystals of greater dimensions or in the case of (ii) the melting of the solid phase obtained after the previous transition. Lee et al.11 observed a small endothermic peak prior to melting of TDF form B (but not for TDF form I) and have attributed this peak to the conversion of form B to form I; but in a second paper,12 the authors have proposed that this transition does not occur and that these small endotherms appeared before melting in the DSC curves obtained for solvates of TDF could be due to intermediate phase transitions or due to the presence of lower-melting point impurities. TOA. According to TOA analysis, TDF melts from 115.2 up to 116.7 °C (Figure 4), which is in agreement with the melting temperatures observed in DSC experiments. The irregular morphology presented by TDF is in accordance with the polymorphic form I.11 FTIR Spectroscopy. The band assignments were performed according to the literature.13 Figure 5 shows an expansion (400−2500 cm−1) of the TDF FTIR spectrum. TDF exhibits a characteristic N−H bending band at 1622 cm−1. The band due to stretching vibration of the imine group (R2−CNR) is observed at 1674 cm−1. An intense band due to primary aromatic amine stretching vibration is observed at 1255 cm−1. The band of carbonyl stretching vibration is observed at 1751 cm−1, and the band due to PO stretching vibration is

observed at 1184 cm−1 (Figure 5). Broad bands due to the stretching vibration of −NH2 and −OH groups are observed at 3200−3500 cm−1 (not shown). XRD. X-ray diffraction measurements show no evidence of any other contaminant phases or clear existence of different polymorphs for this batch. TDF crystallizes under the Monoclinic P21 space group, with two molecules in the asymmetric unit. Fitted lattice parameters obtained by Rietveld analysis are a = 9.835 ± 0.002 Å, b = 22.315 ± 0.005 Å, and c = 12.545 ± 0.003 Å with α = γ = 90.000° and β = 95.042 ± 0.003°. Figure 6 shows the unit cell of TDF. The PXRD pattern of TDF can be seen in Figure 9, which is in accordance with the PXRD pattern of TDF form I.11 ssNMR Spectroscopy. 13C CP-MAS and 1H MAS spectra of TDF are shown in Figure 7. It is straightforward to see that the 13 C CP-MAS spectrum here reported corresponds to that previously ascribed to form I by Lee et al.,11 who, however, did not report any detailed spectral interpretation. A qualitative analysis of the presence of several resonances in the 13C CPMAS spectrum, more than the number of inequivalent carbon nuclei in the molecule, suggests that there is not a single independent molecule in the unit cell of TDF. This is in full agreement with X-ray structural data, which has shown that there are two independent molecules in the asymmetric unit. The 2D MAS-J-HMQC experiment (shown in the Supporting Information) shows correlations between directly bonded carbon−proton couples, while in the NQS spectra (shown in the Supporting Information), signals arising from secondary and tertiary carbons are partially suppressed.

Figure 4. TOA images at 100× magnification of tenofovir disoproxil fumarate. 1917



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Table 1. Assignment of 13C Solid-State Spectrum of Tenofovir Disoproxil Fumarate

Figure 6. Unit cell of the form I of tenofovir disoproxil fumarate.

The assignment of the 13C spectral resonances of TDF to the different nuclei, reported in Table 1, was performed on the basis of CP-MAS, MAS-J-HMQC, and NQS experiments together with semiempirical calculations and comparison with literature data for the fumarate fragment.14 The assignment of the region from 110 to 160 ppm was particularly delicate: in particular, from MAS-J-HMQC experiment (Supporting Information), we can see that signals at 133.2, 141.6, 144.8, 146.6 and 151.1 ppm give rise to correlations with protons, and they are, therefore, ascribable to protonated carbons. On the other hand, NQS experiment (Supporting Information) indicates that the signal at 133.2 and the group of signals at 141.6−146.6 ppm arise from non-quaternary carbons and the signal at 154.2 ppm arises from quaternary carbons (in agreement with MAS-J-HMQC), while the signal at 151.1 ppm could be the superposition of two resonances arising from a quaternary and a non-quaternary carbon. Although the CP experiment is not strictly quantitative, the assignment abovereported is in agreement with a rough evaluation of peak integrals. It is worth noting that all the carbon atoms of the aliphatic portion of the tenofovir disoproxil molecule (except C6 and the carbonyl atoms C10 and C14) present a doublet in the 13C CP spectrum, indicating that the main differences between the two independent molecules in the asymmetric unit of the crystal lattice involve the aliphatic moieties and are probably ascribable to the presence of two different conformations.

carbon number

chemical shift (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

133.2 116.7−117.9 154.2 151.1 151.1 49.2 77.1−79.3 62.2−64.0 84.9−86.8 154.2 72.3−74.2−75.4 21.6−22.8−23.7 84.9−86.8 154.2 72.3−74.2−75.4 21.6−22.8−23.7 21.6−22.8−23.7 21.6−22.8−23.7 15.3−17.0 171.8−172.8 141.6−144.8−146.6 141.6−144.8−146.6 171.8−172.8

The 1H MAS spectrum of TDF, recorded at a spinning rate of 17 kHz, is reported in Figure 7. At this spinning rate, the strong 1H−1H homonuclear dipolar interactions are not completely removed and five main signals can be distinguished. The most intense signal at 1.4 ppm can be assigned to the methyl protons, while the peaks at 5.4 and 8.6 ppm can be ascribed to CH and CH2 aliphatic protons, and aromatic and olefinic protons, respectively. The signals at 17.5 and 21.3 ppm indicate the presence of protons involved in very strong hydrogen bonds. Similar chemical shifts have been observed in some Schiff bases,15 supporting the hypothesis that carboxylic protons form hydrogen bonds with the imminic nitrogens of the rings. Variable-Temperature Studies of TDF. DSC curves of TDF were obtained at different heating rates (Figure 8). As the heating rate increases, an increase in the melting temperature and a broadening of the melting range of TDF are observed because the sample has less time to absorb heat and melt homogeneously at a constant temperature. Instead, the heating device may have warmed well above the real melting range

Figure 7. 13C CP-MAS (left) and 1H MAS (right) spectra of tenofovir disoproxil fumarate recorded at a MAS frequency of 17 kHz. 1918



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Figure 8. DSC curves of tenofovir disoproxil fumarate at different heating rates under a dynamic atmosphere of nitrogen: (A) 2, (B) 10 and (C) 20 °C min−1.

Table 2. Dependence of the Heating Rate on the Melting Range and Heat of Fusion of Tenofovir Disoproxil Fumarate heating rate (°C min−1)

onset temperature (°C)

peak temperature (°C)

endset temperature (°C)

melting range (°C)

heat of fusion (J g−1)

2 10 20

112.8 113.5 114.4

114.0 115.4 116.9

115.0 117.8 120.6

2.2 4.3 6.2

134.93 129.11 107.22

Figure 9. PXRD patterns of tenofovir disoproxil fumarate before (A) and after heating treatment up to 109 °C (B) and 110 °C (C). The peaks that are present only in the PXRD pattern of the sample submitted to a heating up to 109 °C are marked as asterisks.

contrary to α-D-glucose, the heat of fusion of TDF melting decreases with increasing heating rate (Table 2). DSC curves recorded at 10 and 20 °C min−1 show a broad endothermic peak before melting. As pointed out before, this peak can be attributed to (i) melting of very small particles or to (ii) a solid−solid phase transition involving the whole

before the interior liquefies, so the observed range may be at higher temperatures. The superheating of crystalline TDF is observed as shifting the melting peak to higher temperatures at higher heating rates, above the equilibrium melting temperature due to the slow melting process. A similar phenomenon was observed for the melting temperature of α-D-glucose,16 but 1919



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in the two molecules of the asymmetric unit after heating. This is in agreement with the alteration observed in the signals of C9 and C13, in the phosphonate vicinity. The 31P MAS solid-state NMR spectra of TDF confirm the lack of equivalence of the P atoms in the unit cell after heating treatment (Figure 11). The signal observed in the spectrum recorded at room temperature transforms into a doublet during heating up to 80 °C. The spectra reported in Figure 11 show that the phase transition follows a quite slow kinetics: a second spectrum recorded at 80 °C 2 h after the first presents indeed significant differences. In particular, the peak at 0.8 ppm, characteristic of form I, is still quite intense in the first spectrum at 80 °C, while it has almost completely disappeared in the second one, indicating that the phase transition keeps proceeding over 2 h. On the other hand, 1 H MAS spectra do not show any change associated with phase transition, probably because of the small chemical shift dispersion of 1H nuclei, and to the low spectral resolution. All the changes in 13C and 31P spectra with temperature indicate that Form I undergoes a solid−solid transition upon heating, which could imply differences in the crystal packing and/or in the conformation of TDF leading to either a packing or a conformational polymorph. From the spectra, it is clear that this transition involves the whole sample, even if with a slow kinetics. Figure 12 shows the differences in the crystal lattice (obtained from PXRD data) of the sample heated up to 110 °C (Figure 12B), when compared with the sample at room temperature (Figure 12A). Before heating, there is a “sandwichtype” structure formed by two parallel layers, each one formed by two molecules (one up and one down) from each side of the unit cell. The 8 molecules in the unit cell form two “sandwich structures” with a channel between them; each one is composed by two layers of two molecules each, giving a total of four molecules per “sandwich”. After heating, there is a more disorganized structure. Furthermore, we can see that the conformation of the fumarate portion of the molecules changes significantly in the unit cell. This result is in agreement with the changes in the signals related to the carbon atoms of the fumarate moiety in the NMR spectra. The carbonyl carbons of the carboxylic acid functional groups in the fumarate structure (C20 and C23) resonate at 172.7 ppm, and after heating, this signal is no longer single and the presence of a shoulder is clearly observed. This suggests the loss of symmetry of the two fumarate molecules in the asymmetric unit of the crystal lattice after heating. Additional changes in the spectra obtained during heating are observed in the signals related to the methyl carbons C12, C16, C17, and C18, which resonate between 21.5 and 23.7 ppm, and in the signals related to the ternary carbon bonded to them (C11 and C15), which resonate between 72.3 and 75.3 ppm. The two groups of signals related to methyl carbons C12, C16, C17, and C18, and methine carbons C11 and C15 show a narrower chemical shift dispersion after heating, suggesting a larger similarity of the chemical environments in this terminal region between the two inequivalent molecules and/or the two aliphatic chains in the same molecule. Finally, the changes observed in the NMR spectrum after heating of TDF are preserved even when the sample is cooled back (spectrum labeled “30 °C back” in Figures 10 and 11). By comparing PXRD and ssNMR results, we can infer that there is a solid−solid phase transition, and therefore, hypothesis (ii) is supported. We will name the form obtained by heating, which is stable after subsequent cooling, as Form I-1 since it has

sample. The shift toward higher temperature with increasing the heating rate is observed for both peaks present in each curve, and the temperature difference between the two peaks increases as well. The DSC curve obtained at a heating rate of 2 °C min−1 does not show the broad endotherm before melting, which could be due to the fact that the first endothermic peak is very broad and/or partially superimposed to the second endothermic peak. The DSC curve obtained at 10 °C min−1 shows the first endothermic peak at 107 °C. In the curve obtained with the heating rate of 20 °C min−1, the first endothermic peak is at 115 °C. The sample was submitted to a heating treatment up to 109 °C at 10 °C min−1 and subsequently cooled to room temperature and heated again from room temperature up to 150 °C at 10 °C min−1. The DSC curve of the first heating shows the broad endothermic peak (Tonset 107 °C) before TDF melting, but the DSC curve of the second run does not exhibit this broad peak. This observation supports the hypothesis (ii) if the form obtained after the phase transition is stable even when the sample returns to room temperature. The PXRD pattern obtained for the sample after the heating treatment up to 109 °C (Figure 9B) exhibits new reflections peaks (or much more intense) at 6, 8.5, and 12° (2θ). This result could be explained as follows: the temperature is not high enough so that the phase transition can occur completely, but is sufficient to initiate the process. Therefore, there are extra reflection planes due to an intermediate state between the two phases. When the sample is submitted to a heating treatment up to 110 °C, the PXRD pattern does not show these extra reflection peaks (Figure 9C), probably because the phase transition has already occurred and there is no longer an intermediate condition which allows observation of extra Bragg reflections peaks present in the PXRD of the sample submitted to a heating up to 109 °C. The peaks in the PXRD pattern obtained after heating up to 110 °C are essentially at the same positions compared with the PXRD pattern obtained for the sample at room temperature (Figure 9A), which indicates that there is not a significant change in lattice parameters. It means that there is not a structural phase transition of TDF upon heating; i.e., the symmetry and stoichiometry inside the lattice are preserved (Table 3), Table 3. Lattice Parameters of Tenofovir Disoproxil Fumarate Obtained at Room Temperature and after Heating Treatment up to 110 °C parameter

TDF at room temperature

TDF at 110 °C

change in lattice parameter (%)

a (Å) b (Å) c (Å)

18.6105 34.4638 10.0214

18.4312 33.9371 9.8638

−0.96 −1.53 −1.57

contrary to what was observed with the TDF form B-MeOH, which presents a phenomenon of anisotropic lattice contraction/expansion induced by thermal changes.11 In Table 3, we can see that the unit cell length decreases in all three axis, but within the experimental error, and it is not a significant change. The variable-temperature 13C CP-MAS NMR spectra of the drug, recorded from 21 to 80 °C (nominal temperature), are shown in Figure 10. The signals related to the carbon atom of the phosphonate group (C8) show significant changes. Before heating, a doublet is observed due to the C−P scalar coupling (1J ≈ 170 Hz). After heating, this doublet changes into a double doublet, probably due to the lack of equivalence of this region 1920



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Figure 10. 13C CP-MAS solid-state NMR spectra of tenofovir disoproxil fumarate under heating.

Figure 11. Variable-temperature 1H (left) and 31P (right) MAS solidstate NMR spectra of tenofovir disoproxil fumarate recorded at a spinning frequency of 17 kHz.

Figure 12. Differences in crystal lattice of tenofovir disoproxil fumarate before (A) and after (B) heating treatment up to 110 °C.

many characteristics in common with Form I. Consequently, we can safely assign the endothermic peak observed in DSC curves at about 107 °C to the I → I-1 solid−solid transition. As indicated by NMR, this transition involves the whole sample, and a simultaneous change in the crystallites size cannot be

ruled out. Furthermore, the absence of the endothermic peak at 107 °C in the second DSC heating run indicates that form I-1 remains stable after cooling back the sample to room temperature, as observed also in the NMR experiments. 1921



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Two recent examples of similar polymorphism of different organic crystals are reported in refs17 and18. Moreover, we can infer that the observed transition is of the conformational type,19 since a structural phase transition can be ruled out by the analysis of PXRD spectra. It must be noticed that PXRD analyses were obtained after heating up to 110 °C because this is the temperature at which the small endotherm is observed in the DSC curve. ssNMR experiments were conducted up to a nominal temperature of 80 °C, and the phase transition is observed. In the case of ssNMR, we must consider that the real temperature of the sample is higher than the nominal one because of the frictional heating caused by a quite high spinning rate (17 kHz). At this spinning frequency, a discrepancy of about 15 °C between the nominal and the real temperature is expected on the basis of temperature calibration procedures performed on lead nitrate.20 Moreover, ssNMR experiments showed that the observed phenomenon has a slow kinetics, and therefore, the heating rate can dramatically affect its temperature. It is worth noticing that ssNMR spectra were conducted at regular intervals of 10 °C and each experiment lasted about 2 h, while PXRD data were obtained after heating up directly to 110 °C. All of these considerations can explain the temperature differences observed by DSC, PXRD, and ssNMR for the conformational transition.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +55 31 92791609 (E.C.d.L.G.). *E-mail: [email protected]. Tel: +55 31 34096376. Fax: +55 31 34095700 (M.I.Y.). Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to CNPq, FAPEMIG, and UFMG for providing financial assistance. ABBREVIATIONS TDF, tenofovir disoproxil fumarate; TG, thermogravimetry; DSC, differential scanning calorimetry; ssNMR, solid-state nuclear magnetic resonance

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REFERENCES

(1) Gallo, R. C.; Sarin, P. S.; Gelmann, E. P.; Robert-guroff, M.; Richardson, E.; Kalyanaramamv, S.; Man, D.; Sidhu, G. D.; Stahlr, E.; Zolla-Pazzne, S.; Leibowitch, J.; Popovic, M. Science 1983, 220, 865− 867. (2) Barre-Sinoussi, F.; Chermannj, C.; Rey, F.; Nugeyre, M. T.; Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.; Vezinet-Brun, F.; Rozinoux, C.; Rezenbaum, W.; Montaignier, L. Science 1983, 220, 868−871. (3) Gallant, J. E.; Deresinski, S. Clin. Infect. Dis. 2003, 37, 944−950. (4) Chapman, T.; Mcgavin, J.; Noble, S. Drugs 2003, 63, 1597−1608. (5) http://www.who.int/medicines/publications/EssMedList15.pdf (accessed in March 2015). (6) http://www.who.int/hiv/pub/guidelines/artadultguidelines.pdf (accessed in March 2015). (7) http://www.who.int/hiv/pub/guidelines/paediatric020907.pdf (accessed in March 2015). (8) Pines, A.; Gibby, M. G.; Waugh, J. S. J. Chem. Phys. 1973, 59, 569−590. (9) Fung, B. M.; Khitrin, A. K.; Ermolaev, K. J. Magn. Reson. 2000, 142, 97−101. (10) Lesage, A.; Sakellariou, D.; Steuernagel, S.; Emsley, L. J. Am. Chem. Soc. 1998, 120, 13194−13201. (11) Lee, E. H.; Smith, D. T.; Fanwick, P. E.; Byrn, S. R. Cryst. Growth Des. 2010, 10, 2314−2322. (12) Lee, J.; Boerrigter, S. X. M.; Jung, Y. W.; Byun, Y.; Yuk, S. H.; Byrn, S. R.; Lee, E. H. Eur. J. Pharm. Sci. 2013, 50, 253−262. (13) Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric Identification of Organic Compounds, 7th ed.; John Wiley: New York, 2005. (14) Apperley, D. C.; Markwell, A. F.; Harris, R. K.; Hodgkinson, P. Magn. Reson. Chem. 2012, 50, 680−690. (15) Sharif, S.; Schagen, D.; Toney, M. D.; Limbach, H. H. J. Am. Chem. Soc. 2007, 129, 4440−4455. (16) Magón, A.; Pyda, M. Carbohydr. Res. 2011, 346, 2558−2566. (17) Tamboli, M. I.; Krishnaswamy, S.; Gonnade, R. G.; Shashidhar, M. S. Cryst. Growth Des. 2014, 14, 4985−4996. (18) Wang, W. D.; Gao, X.; Strohmeier, M.; Wang, W.; Bai, S.; Dybowski, C. J. Phys. Chem. B 2012, 116, 3641−3649. (19) Cruz-Cabeza, A. J.; Bernstein, J. Chem. Rev. 2014, 114, 2170− 2191. (20) Bielecki, A.; Burum, D. P. J. Magn. Reson., Ser. A 1995, 116, 215−220.

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CONCLUSIONS Several techniques were used to investigate the structural and dynamic behavior of the polymorphic form I of the antiretroviral drug tenofovir disoproxil fumarate. Many different high-resolution solid-state NMR techniques (MAS-J-HMQC, NQS, etc.) have been applied to provide a full spectral assignment of 13C resonances, necessary for more detailed structural and dynamic knowledge, which was never found in the literature before. The TDF form I structure was investigated with several techniques, such as ssNMR, DSC, and PXRD. The PXRD pattern and the solid-state NMR spectra obtained upon heating show that the main changes observed are in the phosphonate vicinity and the fumarate portion of the molecule. PXRD experiments show no evidence of a structural phase transition of the polymorphic form I of TDF upon heating; i.e., the symmetry and stoichiometry inside the lattice are preserved. On the other hand, ssNMR experiments showed that some changes in the crystal structure occur by heating. The occurrence of a conformational phase transition is compatible with both X-ray and ssNMR data, and it also agrees with DSC results. It was shown that the obtained form (here named Form I-1) is stable after subsequent cooling. This study of the structural and dynamic behavior of TDF should represent an important basis of knowledge for understanding the links between the molecular behavior and the possible interactions of TDF with other drugs and excipients in the development of new pharmaceutical formulations containing TDF.

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ASSOCIATED CONTENT

S Supporting Information *

HMQC spectrum of tenofovir disoproxil fumarate, nonquaternary suppression (NQS) experiments of tenofovir disoproxil fumarate. This material is available free of charge via the Internet at http://pubs.acs.org. 1922


Characterization of Tenofovir Disoproxil Fumarate and Its Behavior

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