Combining Nuclear Magnetic Resonance Spectroscopy and Density Functional Theory Calculations to Characterize Carvedilol Polymorphs

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Combining Nuclear Magnetic Resonance Spectroscopy and Density Functional Theory Calculations to Characterize Carvedilol Polymorphs CARLOS A. REZENDE,1 ROSANE A. S. SAN GIL,2 LEANDRO B. BORRE´ ,2 JOSE´ RICARDO PIRES,3 VIVIANE S. VAISS,4 ˜ 4 RICARDO B. DE ALENCASTRO,2 KATIA Z. LEAL1 JACKSON A. L. C. RESENDE,1 ALEXANDRE A. LEITAO, 1

´ Brazil Universidade Federal Fluminense, Instituto de Qu´ımica,, CEP24020-150, Niteroi, Universidade Federal do Rio de Janeiro, Instituto de Quimica, CEP21941-900, Rio de Janeiro, Brazil 3 Universidade Federal do Rio de Janeiro, Instituto de Bioqu´ımica M´edica, CEP21941-902, Rio de Janeiro, Brazil 4 Universidade Federal de Juiz de Fora, Departamento de Qu´ımica, CEP36036-330, Juiz de Fora, Brazil 2

Received 2 June 2015; revised 29 July 2015; accepted 19 August 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24641 ABSTRACT: The experiments of carvedilol form II, form III and hydrate by 13 C and 15 N cross-polarization magic-angle spinning (CP MAS) are reported. The GIPAW (gauge-including projector-augmented wave) method from DFT (density functional theory) calculations was used to simulate 13 C and 15 N chemical shifts. A very good agreement was found for the comparison between the global results of experimental and calculated nuclear magnetic resonance (NMR) chemical shifts for carvedilol polymorphs. This work aims a comprehensive understanding C 2015 Wiley Periodicals, Inc. and of carvedilol crystalline forms employing solution and solid-state NMR as well as DFT calculations.  the American Pharmacists Association J Pharm Sci Keywords: solid-state NMR; ab initio calculations; crystal structure; polymorphism; spectroscopy; carvedilol

INTRODUCTION Drugs can exist in different crystal structures, a phenomenon known as polymorphism. Polymorphism can alter drug’s physicochemical properties as solubility, stability, density, and melting point, which are essential to ensure effectiveness, safety, and quality of a product. Thus, the knowledge of the solid-state properties is very important in the pharmaceutical field.1–3 Carvedilol(RS)-1-(carbazol-4-yloxy)-3-[[2-(omethoxyphenoxy) ethyl]amino]-2-propanol (CAR) is a nonselective beta blocker that is used against several heart diseases as hypertension and systolic dysfunction after myocardial infarction.4,5 It is administrated as a racemic compound, although the Senantiomer is responsible for the beta-blocker activity.6 It is the only beta blocker agent with the carbazole moiety in its structure (Fig. 1). CAR is practically insoluble in water and its solubility is pH dependent, which limits not only its bioavailability, but also a pharmaceutical formulation in the desired manner.7,8 Concerning carvedilol polymorphism, three anhydrous forms were described in the literature: forms I,9 II,10 and III,11 and one hydrate form.12 CAR II is the unique that was characterized by 13 C solid-state nuclear magnetic resonance (SSNMR) spectroscopy.13 The identification and structural characterization of polymorphs can be performed using a combination of infrared/ Raman spectroscopy, thermal methods, and X-ray diffraction Correspondence to: Rosane San Gil (Telephone: +55-21-39387737; Fax: +5521-39387944; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com. Journal of Pharmaceutical Sciences  C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

techniques.14 SSNMR, in particular cross-polarization (CP), at magic angle spinning (CP MAS) is also a powerful technique to study and characterize amorphous and crystalline pharmaceutical solids.15–17 It is a valuable source of structural information, mainly because of the sensitivity chemical shifts to the structure of molecular crystals.14,18 This is especially important for solid-state forms that can not be crystallized and studied by single-crystal X-ray techniques. In the case of organic molecules that present nitrogen in its structure, CP MAS uses the magnetization of a highly sensitive nucleus that is transferred by CP to the 13 C/15 N nuclei, allowing acquisition of 13 C/15 N NMR spectra usually in a couple of hours. The shape of the CP MAS NMR spectrum (number, position, and intensity of the lines) of a solid depends on the chemical environment of each carbon or nitrogen atom in the sample and therefore represents the fingerprint of this compound, which can, in principle, be used to discriminate among polymorphs and solvates, ionic salt complexes, or cocrystals.19–21 It has been shown in many research areas that much more information on structural and physicochemical characteristics of materials can be obtained if the experimental data are complemented by the prediction of properties obtained through calculations, which have been used to aid in data interpretation NMR for more than a decade.22 Recently, a theory for calculations of NMR parameters in periodic systems was presented and this theory uses the GIPAW (gauge-including projectoraugmented wave) method for the chemical shielding tensors in crystalline solids. It was developed for calculation of density functional theory approximations made to electrons from heavy nuclei (in the form of pseudopotential atoms).23,24 In this work, we present a powerful combination between experimental and theoretical techniques to understand solution and solid NMR spectra and to solve ambiguous NMR chemical Rezende et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Figure 1. Molecular structure of carvedilol.

shifts assignments of 13 C and 15 N nuclei not described before, for the carvedilol form II, form III and hydrate, which can serve as a basis for further studies of structure, molecular mobility, and interactions mapping. Moreover, this is the first time that carvedilol form III and hydrate are characterized by solid-state NMR.

EXPERIMENTAL Sample Preparation A commercial sample of carvedilol form II (98% of purity; SIGMA) was used without further purification. Crystallizations of carvedilol form II, form III and hydrate were performed under different conditions using methanol and ethyl acetate as solvents, respectively.11 Powder X-Ray Diffraction Powder X-ray diffraction (PXRD) was obtained on a D8 diffrac˚ tometer Bruker AXS using Cu K" radiation (8 = 1.5418 A) with a graphite monochromator. The patterns were recorded at a tube voltage of 40 Kv and a tube current of 40 mA, between 5° and 40° in 22, with a step size of 0.02° and a scan rate of 3 s. The program MERCURY (Version 2.4) was used for the calculation of the theoretical powder patterns from single-crystal data. Differential Scanning Calorimetry and Thermogravimetric Analysis Calorimetric measurements and TG analysis were performed using a NETZSCH model STA 449 F3 equipment. For all experiments, the samples were weighed (5 mg) in a hermetic aluminum pan, nitrogen flow rate of 50 mL/min, heating rate of 10°C/min, and temperature range from 25°C to 200°C. Solution-State NMR Deuterated solvent (DMSO-d6 ) was purchased from Cambridge Isotope Laboratories, Inc. The purity of the samples was confirmed through 1 H and 13 C solution NMR. The samples (30 mg) were dissolved in DMSO-d6 . Samples were referenced to DMSO-d6 (1 H at 2.50 ppm vs. TMS and 13 C at 39.51 ppm vs. TMS). 1 H and 13 C solution-state NMR spectra of carvedilol form II, form III and hydrate were collected on a 7.05T Bruker DRX300 spectrometer operating at 300.17 MHz (1 H) and 75.48 MHz (13 C). The 1 H spectra were recorded with 32 scans, Rezende et al., JOURNAL OF PHARMACEUTICAL SCIENCES

2 s recycle delay, 2 s acquisition time, 0.281 Hz digital FID resolution, 32K time domain with 6010 Hz spectral width. The 13 C spectra were recorded with Waltz 16 1 H broadband decoupling, 1024 scans, 2 s relaxation delay, 1 s acquisition time, 0.755 Hz digital FID resolution, 4K time domain, and 18,797 Hz spectral width. All two-dimensional experiments were performed on a 14.1 T Bruker DRX600 spectrometer operating at 600.13 MHz (1 H) and 150.90 MHz (13 C) using the pulse sequences from the Bruker Software Library. TOCSY and COSY spectra were recorded at spectral width of 6 KHz in both F2 and F1 domain; 2K × 256 data points were acquired with eight scans per increment and the recycle delay of 2 s. Data processing was performed on a 2K × 1K data matrix. 1 H–13 C HSQC and 1 H–13 C HMQC spectra were measured over 2K complex points in F2 and 128 increments in F1, collecting eight scans per increment with a relaxation delay of 1.5 s. The spectral widths were 8 and 24 kHz in F2 and F1 dimensions, respectively. Data processing was performed on a 2K × 1K data matrix. Assignment was carried out using the interactive program SPARKY (v3.106, T. D. Goddard and D. G. Kneller, University of California, San Francisco, California). Solid-State NMR 13

C and 15 N CP MAS NMR spectra of Carvedilol form II, form III and hydrate were collected on a 9.4 T WB Bruker Avance III 400 spectrometer operating at Larmor frequencies of 100.3 MHz (for 13 C) and 40.6 MHz (for 15 N); 3.2 mm tripleresonance MAS probe was employed. Samples were spun at 10 KHz in ZrO2 rotors and recorded at room temperature. Highresolution spectra were obtained using CP MAS method. Samples were referenced to glycine (C=O at 176.03 ppm vs. TMS and NH3 at −347.54 ppm vs. CH3 NO2 ). Acquisitions were performed using CP.ramp.100 pulse sequence with 4.5 :s proton 90° pulse and recycle delay of 5 s. All spectra were recorded by using 1 ms (13 C) and 4 ms (15 N) contact times and 512 scans. Data were processed using the software Topspin (v2.0; Bruker BioSpin GmbH, Germany). Density Functional Theory Calculations All the ab initio calculations were performed using the codes available in the Quantum-Espresso package,25 which employs DFT26,27 theory, periodic boundary conditions, and plane wave basis sets. The Kohn–Sham orbitals were expanded in a plane wave basis set with a maximum kinetic energy cutoff of 60 and DOI 10.1002/jps.24641

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

480 Ry for the charge density. We adopted the generalized gradient approximation GGA-PBE28 for the exchange–correlation functional and projector-augmented wave29 method to describe the interaction of valence electrons with nuclei and core electrons. Monkhorst and Pack30 meshes of 1 × 1 × 1 k-point sampling in the first Brillouin zone were used for all cells. The projector-augmented waves used for the C, O, N, and H atoms were created from files available in the pslibrary.0.3.1.31 The initial structures from CAR II10 and III11 used in the calculations were taken from the Cambridge Structural Data Base,3 2 whose unit cells have 224 atoms, belongs to the space group P21 /c (CAR II) and P21 /n (CAR III), and have the fol˚ b = 15.2050 A, ˚ c= lowing lattice parameters: a = 15.5414 A; ˚ " = 90°, $ = 100.73°, and ( = 90° for CAR II and a = 9.1174 A, ˚ b = 12.2293 A, ˚ c = 14.9933 A, ˚ " = 90°, $ = 91.39°, 11.2908 A, and ( = 90° for CAR III. Two different optimization procedures were used: in the first, the atomic positions for all structures were optimized (lattice parameters were kept fixed equal to experimental values). In the second, only the hydrogen atoms were optimized (heavy atoms were kept fixed). The relative ion positions were relaxed until all of the force components were smaller than 0.001 Ry/bohr. The absolute chemical shielding tensors for each nucleus Fiso (r) were calculated using GIPAW23,24 method. The conversion of the calculated isotropic chemical shielding, Fiso = Tr[F/3], into the corresponding isotropic chemical shifts (*iso ) was performed by the expression: *iso = (Fiso − Fref )/m, where Fref and m were determined by fitting Fiso to the measured chemical shifts (*exp ) by means of a linear regression (Fref and m are the intercept and slope of the regression model, respectively).22

RESULTS AND DISCUSSION Powder X-Ray Diffraction Three PXRD from carvedilol form II, form III and hydrate were analyzed by PXRD (Fig. S1, Supplementary Material). The PXRD patterns showed sharp diffraction peaks, indicating their crystalline nature. The experimental data were compared with the pattern simulated from the single-crystal structures previously published and the high crystallographic purity of polymorphs II and III and hydrate could be confirmed.11 The minor differences between experimental and calculated data can be attributed to preferred orientation and thermal contraction. Differential Scanning Calorimetry and Thermogravimetry Thermal methods can be used to clearly distinguish between polymorphs and solvates, during desolvation of the latter.33 differential scanning calorimetry (DSC) and thermogravimetric analysis were carried out to confirm carvedilol polymorphs and the hydrate form (Fig. S2, Supplementary Material). Carvedilol forms II, III, and hydrate DSC show one endotermic peak at 119°C, 127°C, and 109°C for each form, respectively. Furthermore, TG analysis showed mass loss around 2.2% in the hydrate and no mass loss for carvedilol form II and form III. These results are in agreement with data previously published.11 Solution-State NMR 1

H solution spectra were acquired for carvedilol form II, form III and hydrate. As it can be clearly seen, they are identical, DOI 10.1002/jps.24641

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confirming that the same basic molecular structure of CAR is present in the three forms and the absence of impurities. In solution, rapid isotropic tumbling averages the anisotropic provides the same chemical shift values for crystal packing polymorphs as forms II, III, and hydrate (Fig. 2). The 1 H and 13 C chemical shifts assignments were established through TOCSY, COSY, 1 H–13 C HSQC (Fig. S3a, Supplementary Material) and 1 H–13 C HMBC experiments (Fig. S3b, Supplementary Material). By comparing our results with data from literature,34–36 it would be possible to state that the 13 C chemical shifts are similar, but the 1 H assignments of H19, H20, H21, and H22 are different compared with data published by Zielinska-Pislak et al.34 (Table 1).

Table 1. Solution-State NMR—1 H and 13 C Carvedilol Chemical Shifts (DMSO-d6 ) Atom

This Work Chemical Shifts (ppm)

Ref. 34 Chemical Shifts (ppm)

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 H2 H3 H4 H7 H8 H9 H10 H13 H14 H15 H16 H17 H19 H20 H21 H22 H24 HN1 HN2 OH

154.6 100.1 126.0 103.6 140.9 138.7 110.1 124.1 118.2 122.2 121.6 111.4 70.2 68.3 52.3 48.3 68.3 147.9 113.3 120.5 120.7 112.0 149.0 55.1 6.70–d 7.30–t 7.09–d 7.46–d 7.35–t 7.15–t 8.25–d 4.19–m 4.15–m 2.85–m 2.95–t 4.02–t 6.94–m 6.84–m 6.87–m 6.94–m 3.73–s 2.02 11.25 5.19

156.0 100.4 126.5 103.8 141.1 138.9 110.3 124.5 118.6 122.5 121.8 111.6 70.5 68.4 52.6 48.5 68.5 148.1 113.7 120.7 121.0 112.2 149.2 55.4 6.69–d 7.29–t 7.08–d 7.64–d 7.34–t 7.14–t 8.25–d 4.17–m 4.17–m 2.89–m 2.94–m 4.01–t 6.86–m 6.92–m 6.92–m 6.86–m 3.72–s 2.01 11.26 5.19

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Figure 2.

1H

NMR spectra (300 MHz, DMSO-d6 ) of carvedilol form II, form III and hydrate.

Solid-State NMR Carvedilol(RS)-1-(carbazol-4-yloxy)-3-[[2-(omethoxyphenoxy) ethyl]amino]-2-propanol forms II and III crystallize in the centrosymmetric monoclinic space group with four molecules (comprising two pairs of enantiomeric molecules) per unit cell11 and the hydrate form contains two independents carvedilol molecules (molecules A and B) and one water molecule in the asymmetric unit. 13 C and 15 N CP MAS NMR spectra obtained for Carvedilol form II, form III and hydrate are depicted in Figures 3 and 4, respectively. The narrow shapes of the signals in the spectrum of CAR II, when compared with the polymorph III and hydrate form, indicate that CAR II presents the higher crystallinity. The 13 C assignments of CAR II (Table 2) were established by comparing the experimental data with the 13 C chemical shifts calculations and the 13 C data reported by Zielinska-Pislak et al.13 13 C CP MAS NMR spectra consist of 24 well-resolved resonances divided in two distinct domains: the aliphatic carbons (C13–C17 and C24) appear between 49 and 73 ppm, and the aromatic carbons (C1–12 and C18–23), which correspond to two different motifs (carbazol-4-yloxy and 2-methoxyphenoxy), appear between 101 and 155 ppm. 15 N CP MAS NMR spectrum consists of two well-resolved resonances at −348.2 ppm (N1) and −266.4 ppm (N2) and was reported in the first time here (Fig. 4; Table 2). For CAR III, the 13 C assignments (Table 2) were established comparing the 13 C chemical shifts from CAR II and 13 C chemical shifts calculations. 13 C CP MAS NMR spectra consist of 22 well-resolved resonances, two ambiguous peaks centered at 120.3 ppm (C9 and C10) and at 121.4 ppm (C11 and C20). 15 N CP MAS NMR spectrum consists of two well-resolved resonances at −347.8 ppm (N1) and −264.6 ppm (N2) (Fig. 4; Table 2). CAR II and CAR III show a typical case of conformational polymorphism11 and the Rezende et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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C CP MAS data suggest that this technique can distinguish active pharmaceutical ingredients with conformational polymorphism properties. The 13 C assignments of hydrate form (Table 2) were established by comparing the 13 C chemical shifts from CAR II and III. The 13 C CP MAS NMR spectra consist of 38 well-resolved resonances, and for carbons 1, 2, 4, 13–17, 20, and 24, the signals are duplicated. The large number of peaks in the 13 C CP MAS NMR spectra compared with CAR II and CAR III ones would be because of different orientations of the carbazol-4-yloxy, aliphatic central chain, and 2 methoxyphenoxy moieties in the distinct molecules A and B. 15 N CP MAS NMR spectra consist of four wellresolved resonances, −350.8 ppm (N1A), −347.0 ppm (N1B), −269.2 ppm (N2B), and −263.6 ppm (N2A) (Fig. 4; Table 2). These two resonances for each nitrogen atom occur because the carvedilol molecules A and B are not similar, and both of them are involved in hydrogen bonds with water molecule and hydroxyl moieties.10 This result suggests that 15 N CP MAS is a powerful technique to distinguish polymorphs and hydrates. Comparing solid-state NMR 13 C chemical shifts from carvedilol polymorphs (Table 2) and 13 C solution-state NMR data (Table 1), it is possible to verify that the assignments from carvedilol in solution state are an average of different structural conformations and the two polymorphs and hydrate form are presented in this work. Chemical Shifts Calculations In the majority of literature data, the assignment of small organic molecules is based on previous solution-state NMR chemical shifts assignments. However, this strategy does not consider possible polymorphs and it could lead to some mistakes during chemical shift assignment. GIPAW method has been DOI 10.1002/jps.24641

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Figure 3.

13 C

CP MAS NMR spectra (100.3 MHz) of carvedilol form II, form III and hydrate.

Figure 4.

15 N

CP MAS NMR spectra (40.6 MHz) of carvedilol form II, form III and hydrate.

DOI 10.1002/jps.24641

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Table 2. 13 C and 15 N Solid-State NMR Chemical Shifts (ppm) of Carvedilol Polymorphs and Hydrate CAR Form II

Atom C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 N1 N2

CAR Form III

CAR Hydrate

Ref. 13 * (ppm)

This Work * (ppm)a

This Work * (ppm)a

This Work * (ppm)a

154.3 100.4 129.5 103.4 139.7 139.7 110.4 119.6 119.6 123.1 122.1 112.1 72.5 69.2 51.5 48.9 70.7 148.1 110.4 118.5 121.4 110.4 149.6 53.5 – –

154.4 (−0.9) 101.0 (1.1) 126.2 (1.9) 103.5 (1.1) 140.0 (1.5) 139.3 (1.2) 110.6 (1.0) 129.7 (0.7) 118.8 (−0.3) 121.5 (1.1) 122.2 (−0.1) 112.3 (0.6) 70.9 (−0.3) 69.2 (−1.3) 52.0 (−0.2) 49.2 (0.3) 72.7 (−1.7) 148.1 (−2.0) 119.4 (0.2) 112.5 (−5.3) 123.3 (0.5) 110.3 (1.4) 149.8 (−1.3) 53.6 (1.1) −348.2 −266.4

154.6 (−0.7) 100.2 (−0.3) 126.4 (−0.2) 103.9 (1.0) 141.2 (0.9) 139.1 (0.2) 114.5 (0.4) 123.2 (0.7) 120.3 (0.7) 120.3 (−0.3) 121.4 (−0.5) 111.5 (−0.9) 69.4 (−0.4) 69.9 (−1.6) 55.0 (−0.4) 49.8 (0.5) 65.5 (0.2) 147.9 (−0.9) 112.6 (−0.2) 121.4 (0.4) 120.3 (1.0) 109.3 (0.7) 146.9 (−0.7) 53.0 (0.6) −347.8 −264.6

156.4; 154.4 98.6; 97.7 128.4 102.6; 100.9 142.4 139.6 111.2 126.3 122.7 124.8 124.4 112.3 68.9; 68.1 73.0; 71.5 53.7; 50.0 49.0; 47.3 67.3; 66.0 147.7 111.2 120.7; 119.7 123.8 111.2 148.5 55.9; 54.2 −347.0; −350.8 −263.4; −270.0

a The difference between 13 C experimental and calculated NMR chemical shifts from carvedilol form II and form III is in brackets.

widely used to calculate chemical shielding constants of pharmaceutical compounds with very good accuracy.37–42 Regarding carvedilol, which has three polymorphs and other solvates, the 13 C CP MAS spectra of forms II, III, and hydrate show that the compounds are not the same, but they are very similar. The difference between 13 C chemical shifts from solution-state and 13 C solid-state chemical shift (Fig. 5) of carvedilol form II, form III and hydrate is around 1 ppm, and the maximum difference observed is 3.6 ppm for peak 6 at 126.1 ppm of CAR II. Thus, in order to achieve our objective, we have resorted to the GIPAW calculations of 13 C and 15 N chemical shielding constants to validate the assignments and help solve ambiguities, preceded by the crystal structure optimization. For both polymorphs (CAR II and III), 13 C calculated chemical shielding are in good agreement with the experimental data. Experimental chemical shifts versus calculated chemical shielding for 13 C nuclei using QE codes are depicted in Figure 6. In the case of form II, the maximum difference among 13 C calculated and experimental data obtained was 5.3 ppm for an aromatic carbon (C20) (Table 2). However, for the other carbon atoms, this difference is below 2.0 ppm. The results from GIPAW calculations suggest that 11 peaks (C3/C8, C10/C21, C13/C17, C19/C20, and C7/C12/C21) were exchanged compared with data published by Zielinska-Pislak et al.34 where the assignment were performed only comparing liquid- and solid-state NMR data. Therefore, compared with CAR II, the form III presented better results (R2 = 0.999, for structure with all atoms positions Rezende et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Figure 5. Difference between 13 C chemical shifts from solution-state and 13 C solid-state chemical shifts of carvedilol form II, form III, and hydrate.

previous optimization), C14 showed 1.6 ppm of difference between 13 C calculated and experimental NMR chemical shifts and this was the higher difference value obtained (Table 2). An inspection of Figure 6 and Table 2 reveals that performing full geometric optimization did not lead to improvement on the quality of chemical shielding predictions. The coefficient of determination (R2 ) indicates that theoretical model present in this work has high reliability in the calculation of 13 C chemical shifts. 15 N chemical shielding constants were performed by GIPAW method, and the difference between 15 N experimental chemical shift from N1 and N2 (Nexp ) were compared with the difference between 15 N-calculated chemical shielding from the same nitrogen atoms for carvedilol forms II and III (Ncal ). On the contrary, for both polymorphs, independently of how the previous structures optimizations have been performed, the results obtained by 15 N chemical shifts were not as good as for the 13 C chemical shifts. The carvedilol form II shows Nexp = 81.8 ppm and Ncal = 90.3 ppm and the difference between Nexp and Ncal is around 9 ppm. For polymorph III, the absolute values obtained for  were Nexp = 83.2 ppm and Ncal = 96.6 ppm, reaching a difference that exceeds 11 ppm. However, in the carvedilol case, this fact is not a problem because its molecular structure has two distinct atoms of nitrogen (Fig. 1).

CONCLUSION In summary, three forms of carvedilol were studied: polymorphs (II and III) and hydrate. The full assignment of the NMR spectra of carvedilol was performed using a combination of SSNMR techniques and ab initio calculations of 13 C and 15 N chemical shifts with the GIPAW modules of the Quantum-Espresso. Our results suggest that SSNMR chemical shifts assignment based on solution-state NMR data could lead to an incorrect chemical shifts assignments. The combination of CPMAS technique and chemical shifts calculations with GIPAW method could be an alternative approach. 13 C and 15 N CPMAS data showed that DOI 10.1002/jps.24641

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Figure 6. Comparison between experimental chemical shifts versus calculated chemical shielding for previous optimization of only hydrogen positions and of all atoms positions.

this technique is excellent to distinguish carvedilol polymorphs and hydrate. This is the first time that carvedilol 15 N chemical shifts were reported. The two methods (previous optimization of only hydrogen positions and of all atoms positions) used for theoretical determination of 13 C and 15 N chemical shifts yield results in good agreement with the experimental data (R2 > 0.99), which confirms the good quality of the deposited crystal structures of CAR II and CAR III. This procedure may be extended to other pharmaceutical compound.

ACKNOWLEDGMENTS The authors thank LDRX-UFF for X-ray facilities, LAME-UFF for computational resources, and CNPq, CAPES, and FAPERJ for financial support.

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DOI 10.1002/jps.24641