- Email: [email protected]
Structure Determination of Monohydrated Trifolin (Kaempferol 3-O-β-D-Galactopyranoside) from Laboratory Powder Diffraction Data
Structure Determination of Monohydrated Trifolin (Kaempferol 3-O-β-D-Galactopyranoside) from Laboratory Powder Diffraction Data 4 ´ DA SILVA,1,2 JESUS ´ G. D´IAZ,3 JAVIER GONZALEZ-PLATAS ´ IVAN 1
SpLine Spanish CRG Beamline at the ESRF. 6, Rue Jules Horowitz, BP 220, 38043 Grenoble Cedex 09, France
2
Instituto de Ciencia de Materiales de Madrid-ICMM/CSIC, Cantoblanco Madrid 28049, Spain
3
Departamento de Qu´ımica, Universidad de La Laguna. Instituto Universitario de Bio-Org´anica “Antonio Gonz´alez”, 38206 La Laguna, Tenerife, Spain
4 ´ de Rayos X, Universidad de La Laguna, 38206 La Laguna, Departamento de F´ısica Fundamental II, Servicio de Difraccion Tenerife, Spain
Received 14 June 2010; revised 24 September 2010; accepted 24 September 2010 Published online 24 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22379 ABSTRACT: The crystal structure of monohydrated trifolin (kaempferol 3-O-$-Dgalactopyranoside) (an important biologically active compound, which was isolated from the aerial part of Consolida oliveriana) has been determined from conventional laboratory X-ray powder diffraction data. Variable counting time technique was used during measurement and crystal structure was solved by means of Monte Carlo algorithm. The final structure was achieved by Rietveld refinement using both constraints and restraints on interatomic bond lengths and angles. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1588–1593, 2011 Keywords: X-ray powder diffractometry; crystal structure; crystallography; ab initio calculations; Monte Carlo
INTRODUCTION The naturally originated compounds belonging to the group of flavonoids, which are widely distributed in the human diet,1 have generated particular interest with regard to human health effects, including antioxidant activities2,3 or protection of cardiovascular diseases,4–6 and they have been a subject of intensive pharmacological studies in recent years, as they are among the most promising anticancer agents.7–9 In this context, some flavonoid complexes have been previously investigated10 (kaempferol, quercetin, trifolin, hyperoside, 2 -acetylhyperoside, 6 -acetylhyperoside, 7glucotrifolin, biorobin, or robinin). Monohydrated trifolin (C21 H20 O11 ·H2 O) is a derivative of flavonoid and, recently, it has been shown that the acetyl derivate presents cytotoxic properties.11 No suitable crystals ´ da Silva (Tel: +33-476-88-2449; Fax: Correspondence to: Ivan +33-476-88-2816; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 1588–1593 (2011) © 2010 Wiley-Liss, Inc. and the American Pharmacists Association
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for single-crystal diffraction were obtained, but the structural information could be determined from powder diffraction data. Here, we report the ab initio structural determination of a monohydrated trifolin, using a Monte Carlo/ parallel tempering method, in order to obtain a starting model, followed by Rietveld refinement using constraints and restraints on bond lengths and angles.
EXPERIMENTAL Monohydrated trifolin was isolated from Consolida oliveriana. Details of extraction and isolation for the compound have been published previously.10 The sample was microcrystalline and attempts to grow single crystals were unsuccessful. Spectroscopic Study The structural identity of the studied compound (see, Fig. 1) was determined spectroscopically (proton magnetic resonance and 13 C nuclear magnetic resonance, infrared and ultraviolet-visible spectroscopy,
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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program13 ; the first 25 peaks, up to 29◦ in 22, were selected. Pattern indexing was performed with DicVOL06 program14 and a solution was obtained, which yielded a monoclinic cell with figures of merit15,16 of M(25) = 34.1, F(25) = 86.2 (0.0063, 46). For space group determination, Expo2004 program17 was used, where a statistical algorithm to determine the most probable space group have been implemented.18 In this case, the most probable extinction group was P 1 21 1, with a probability factor of 0.776. There are two compatible space groups (P21 and P21 /m) for this extinction group, but taking into account the cell and molecule volumes, they allowed us to choose only one (P21 ). Structure Determination by Monte Carlo Methods
Figure 1. Molecular structure of trifolin.
and mass spectrometry). The details of this study can be found elsewhere.10 X-Ray Diffraction The powder diffraction data were collected on a PANalytical X’Pert Pro diffractometer (PANalytical, Almelo, Netherlands) using Cu-K"1 radiation with a hybrid monochromator for parallel beam (Debye– Scherrer geometry-transmission mode). The sample was introduced in a Hilgenberg glass capillary (diameter = 0.3 mm) and spinning rotation was active to reduce the effect of possible preferential orientations. The detector was a X’Celerator (PANalytical, Almelo. Netherlands), working in continuous mode with 2.149◦ active length; soller slits of 0.02 radians were placed into the incident and diffracted beam path to reduce the asymmetry of peaks due to axial divergence. The 22 range was 3.5◦ –83◦ , with a step width of 0.0169◦ , and the total time of measurement was about 19 h. Variable counting time (VCT) procedure12 was used during data collection; it means that measurement time is increased toward higher 22 angles and it allows to get substantial amount of peaks information present also at high 22 angles, which is otherwise lost in conventional (i.e., fixed counting time) measurements. It is known that VCT procedure, in general, improves results on structure determination and more stable refinement of atoms coordinates and thermal parameters, especially for the organic compounds, are achieved.
RESULTS Data Reduction and Indexing Reflection positions were determined using the peak search algorithm implemented in the WinPLOTR DOI 10.1002/jps
Structure solution, by means of Direct Methods, was attempted with Expo2004 program, within P21 space group, without success. Thus, to obtain a starting structural model, Monte Carlo calculations were performed, using the parallel tempering algorithm implemented in the program FOX.19 A template of the trifolin molecule was built with the software package ChemBio Office (version 11.0), which was introduced in FOX, as well as a water oxygen. During the calculations, the observed and calculated intensities were compared only in the 22 range of 4◦ –52◦ and the molecule could translate and rotate randomly; torsion angles between the benzopyran and phenyl rings and between both benzopyran and glucose moiety and the oxygen atom connecting the two groups could also change, as well as the torsion angle C-OH in the glucose moiety. After ca. 13 million trials, the agreement factors were Rwp = 0.0694 and GoF = 10.054. Rietveld Refinement The solution found by FOX program was introduced in FullProf program20 to perform a Rietveld refinement. Atomic coordinates of the 33 independent nonH atoms were fitted, but constraints on benzopyran and phenyl rings and restraints on the other bond lengths and angles were introduced to limit the number of free parameters and ensure the convergence of the refinement process. The values for these bond lengths and angles were taken from similar molecules in The Cambridge Crystallographic Data Centre (CCDC) database and the mean-square deviations of assigned values were 0.01Å and 1◦ , respectively. Three different isotropic temperature factors were introduced: one for the water oxygen, other for the OH oxygen, and one for the rest of atoms. Intensities were corrected for absorption effects for a cylindrical sample. The peak function used was the Thompson— Cox–Hastings pseudo-Voigt,21 which can take into account the experimental resolution and the broadening JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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Figure 2. Comparison between the observed (red circles) and calculated (black continuous line, upper part) patterns of C21 H20 O11 ·H2 O. The difference curve (blue continuous line, lower part) and the reflection positions (green vertical lines) are also represented in the lower part of the figure.
due to size and strain effects, typical in this type of organic powder samples. The Finger’s treatment of the axial divergence22 was taken into account to model Table 1. Crystal Data and Structure Refinement for C21H20O11·H2O. Crystal data Formula Formula weight Cell setting, space group Temperature (K) a, b, c (Å); $ (◦ ) Volume (Å3 ) Z, Dc (g·cm−3 ) Radiation type : (mm−1 ) Specimen form, color Refinement Refinement method RF , RBRAGG Goodness-of-fit Wavelength (Å) Profile function No. of profile data steps No. of contributing reflections No. of bond length restraints No. of bond angle restraints
C21 H20 O11 ·H2 O 466.39 Monoclinic, P21 298 15.6461(3), 13.7636(2), 4.64731(8); 97.653(2) 991.87(3) 2, 1.56155 Cu K"1 1.010 Cylinder, white Rietveld refinement 0.0525, 0.0331 1.855 1.54056 Thompson–Cox–Hasting pseudo-Voigt 4671 693 25 41
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the asymmetry of the peak profile, and 69 points were chosen regularly distributed on the experimental pattern, in the 22 range 4–83◦ , to model the background through a linear interpolation made between two successive points. Hydrogen atoms for trifolin molecule were introduced in FullProf at their calculated positions with CALC-OH23 program, which combines geometric and force-field calculations on the basis of hydrogenbonding interactions, whereas H atoms’ positions for the water molecule were obtained with CCDC Mercury program.24 On the final Rietveld fit, there are 85 adjustable parameters (scale factor, zero shift, atomic coordinates, temperature factors, cell parameters, and peak shape), taking into account the introduced constraints. The final Rietveld agreement factors were Rp = 0.036, Rwp = 0.047, and χ2 = 3.44. In Figure 2, the plot of the final fit is given. Crystallographic and refinement-related data are reported in Table 1, whereas atomic coordinates and displacement parameters are reported in Table 2.
DISCUSSION The Oak Ridge Thermal Ellipsoid Plot Program drawing of the molecule monohydrated trifolin is shown in DOI 10.1002/jps
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Table 2. Atomic Coordinates and Isotropic Displacement Parameters (Å2) Obtained from Rietveld Refinement. Atom O1w O1 O6 O7 C1’’ C2 C2’’ C3 C3’’ C4 C4’’ C5’’ C6’’ H1’’ H2’’ H3’’ H4’’ H5’’ O2 O3 O4 O5 O8 O9 O10 O11 H3O H4O H5O H8O H9O H10O H11O H1OWa H1OWb H6’’a H6’’b C5 C6 C7 C8 C9 C10 H6 H8 C1’ C2’ C3’ C4’ C5’ C6’ H2’ H3’ H5’ H6’
x
y
z
Uiso
0.2232(10) 0.4065(6) 0.2577(8) 0.1151(5) 0.1888(5) 0.3267(8) 0.1661(6) 0.3220(12) 0.0975(6) 0.3778(10) 0.0184(8) 0.0436(5) −0.0310(9) 0.1987(5) 0.1493(6) 0.1034(6) −0.0334(8) 0.0536(5) 0.36275 0.4902(11) 0.6759(7) 0.1726(10) 0.2433(8) 0.0748(10) 0.0013(11) −0.0155(12) 0.4465(11) 0.6995(7) 0.1277(10) 0.2368(8) 0.1121(10) −0.0392(11) −0.0089(12) 0.246(13) 0.178(10) −0.0432(9) −0.0878(9) 0.51080 0.58150 0.59659 0.54099 0.47009 0.45490 0.61930 0.55099 0.29438 0.21220 0.16708 0.20349 0.28529 0.33109 0.18731 0.11210 0.30989 0.38579
0.83729 0.2176(11) 0.4151(13) 0.3993(10) 0.4489(6) 0.2665(8) 0.5552(6) 0.3605(7) 0.6016(6) 0.4106(8) 0.5455(6) 0.4384(6) 0.3715(6) 0.4376(6) 0.5611(6) 0.6007(6) 0.5588(6) 0.4315(6) 0.49713 0.4716(9) 0.1932(12) 0.0154(12) 0.6052(13) 0.6956(8) 0.5418(14) 0.2729(7) 0.4931(9) 0.2328(12) 0.0374(12) 0.6503(13) 0.7331(8) 0.5042(14) 0.2721(7) 0.819(15) 0.791(12) 0.3726(6) 0.3967(6) 0.38080 0.32771 0.23601 0.19892 0.25241 0.34350 0.35340 0.13752 0.20570 0.22980 0.16790 0.08061 0.05601 0.11731 0.28799 0.18480 −0.00218 0.09991
0.389(3) 0.311(3) 0.147(2) 0.163(3) 0.287(3) 0.243(3) 0.201(3) 0.315(4) 0.366(3) 0.572(4) 0.225(2) 0.279(3) 0.161(4) 0.497(3) −0.009(3) 0.578(3) 0.316(2) 0.491(3) 0.62870 1.045(3) 1.036(4) −0.663(3) 0.292(4) 0.256(4) −0.0800(19) 0.251(4) 0.950(3) 1.152(4) −0.750(3) 0.403(4) 0.331(4) −0.1196(19) 0.429(4) 0.21(3) 0.43(5) −0.074(4) 0.249(4) 0.92260 1.04060 0.93300 0.70469 0.58069 0.69119 1.19301 0.63278 −0.03210 −0.16251 −0.36252 −0.43372 −0.30471 −0.10300 −0.11510 −0.44972 −0.35361 −0.01520
0.092(9) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.061(2) 0.061(2) 0.061(2) 0.061(2) 0.061(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.138(12) 0.138(12) 0.061(2) 0.061(2) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.061(2) 0.061(2) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.061(2) 0.061(2) 0.061(2) 0.061(2)
Figure 3. The trifolin molecule consists of a benzopyran moiety, almost coplanar [0.71(15)◦ as dihedral angle between planes of benzene and pyran rings], a planar phenyl ring rotated by 10.68(13)◦ from the plane of the benzopyran ring system and a galactopyDOI 10.1002/jps
Figure 3. Oak Ridge Thermal Ellipsoid Plot Program drawing of the molecule trifolin in the asymmetric unit with labeling scheme
Figure 4. Molecular packing viewed along c axis.
ranoside ring adopting the 4C1 chair conformation with the benzopyran moiety positioned equatorially as substituent at C1’’. Bond distances and angles are comparable with values reported for similar flavonoids.25 The lengthening of the double bond C4 =O2 [1.238(12) Å] is due to strong intramolecular hydrogen bond between O2 and O3 . JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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Figure 5. Molecular stacking, showing the linking of the molecules by C−H · · · O and B–B interactions.
The hydroxyl group, O3 , has a gauche arrangement with respect to H3 -O3 -C5 -C10 torsion angle (1.74◦ ), giving rise to a short (1.831 Å) intramolecular contact between the H atom of the hydroxyl group and carbonyl atom O2 . The projection of the crystal structure down the c axis is shown in Figure 4. Weak intermolecular interactions play a decisive role in determining the three-dimensional structure for this compound, with the presence of short contacts of C−H · · · O type and B–B interactions with a centroid–centroid distance of 4.761 Å (see, Fig. 5). Final position and orientation of the water molecule allows us to conclude that the water molecule plays a stabilizing role in the molecular packing (see, Figs. 4 and 5).
CONCLUSION In this study, we have shown the structure determination of monohydrated trifolin compound from conventional laboratory X-ray powder diffraction data, using a VCT procedure during measurement. This allowed us to determine the structure by means of Monte Carlo methods and refine the model using the Rietveld method. Recent advances on algorithms aiming to solve crystal structures using powder diffraction data allow us to deal, in an easier way, new structural studies on compounds in which only powder samples can be obtained, as is the case of monohydrated trifolin. CCDC 775168 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data request/cif, or by emailing data request@ccdc. cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
REFERENCES 1. Havsteen BH. 2002. The biochemistry and medical significance of the flavonoids. Pharmacol Ther 96:67–202. 2. Bahorun T, Luximon-Ramma A, Crozier A, Aruoma OI. 2004. Total phenol, flavonoid, proanthocyanidin and vitamin C levels and antioxidant activities of Mauritian vegetables. J Sci Food Agric 84(12):1553–1561. 3. Burns J, Gardner PT, O’Neil J, Crawford S, Morecroft I, McPhail DB. 2000. Relationship among antioxidant activity, vasodilation capacity, and phenolic content of red wines. J Agric Food Chem 48(2):220–230. 4. Engler MB, Engler MM. 2006. The emerging role of flavonoidrich cocoa and chocolate in cardiovascular health and disease. Nutr Rev 64(3):109–118. 5. Sesso HD, Gaziano JM, Liu S, Buring JE. 2003. Flavonoid intake and the risk of cardiovascular disease in women. Am J Clin Nutr 77(6):1400–1408. 6. Yochum L, Kushi LH, Meyer K, Folsom AR. 1999. Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. Am J Epidemiol 149(10):943–949. 7. Middleton E, Kandaswami C, Theoharides TC. 2000. The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751. 8. Ren W, Qiao Z, Wang H, Zhu L, Zhang L. 2003. Flavonoids: Promising anticancer agents. Med Res Rev 23:519–534. 9. Kosmider B, Osiecka R. 2004. Flavonoid compounds: A review of anticancer properties and interactions with cisdiamminedichloroplatinum(II). Drug Dev Res 63(4):200–211. 10. D´ıaz JG, Carmona AJ, Torres F, Quintana J, Est´evez F, Herz W. 2008. Cytotoxic activities of flavonoid glycoside acetates from Consolida oliveriana. Planta Med 74:171–174. 11. Torres F, Quintana J, D´ıaz JG, Carmona AJ, Est´evez F. 2008. Trifolin acetate-induced cell death in human leukemia cells is dependent on caspase-6 and activates the MAPK pathway. Apoptosis 13:716–728. 12. Madsen IC, Hill RJ. 1994. Collection and analysis of powder diffraction data with near-constant counting statistics. J Appl Crystallogr 27:385–392. 13. Roisnel T, Rodriguez-Carvajal J. 2000. WinPLOTR: A windows tool for powder diffraction patterns analysis. Mater Sci Forum 378–381:118–126. 14. Boultif A, Lou¨er D. 2004. Powder pattern indexing with the dichotomy method. J Appl Crystallogr 37:724–731. DOI 10.1002/jps
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15. DeWolff PM. 1968. A simplified criterion for the reliability of a powder pattern indexing. J Appl Crystallogr 1:108– 113. 16. Smith GS, Snyder RL. 1979. FN : A criterion for rating powder diffraction patterns and evaluating the reliability of powder-pattern indexing. J Appl Crystallogr 12:60– 65. 17. Altomare A, Caliandro R, Camalli M, Cuocci C, Giacovazzo C, Moliterni AGG, Rizzi R. 2004. Automatic structure determination from powder data with EXPO2004. J Appl Crystallogr 37:1025–1028. 18. Altomare A, Camalli M, Cuocci C, da Silva I, Giacovazzo C, Moliterni AGG, Rizzi R. 2005. Space group determination: Improvements in EXPO2004. J Appl Crystallogr 38:760– 767. 19. Favre-Nicolin V, Cerny´ R. 2002. FOX, ‘free objects for crystallography’: A modular approach to ab initio structure determination from powder diffraction. J Appl Crystallogr 35:734– 743.
DOI 10.1002/jps
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20. Rodriguez-Carvajal J. 2001. Recent developments of the program FULLPROF. Commission on powder diffraction (IUCr). Newsletter 26:12–19. 21. Thompson P, Cox DE, Hastings JB. 1987. Rietveld refinement of Debye–Scherrer synchrotron X-ray data from A12 03 . J Appl Crystallogr 20:79–83. 22. Finger LW, Cox DE, Jephcoat AP. 1994. A correction for powder diffraction peak asymmetry due to axial divergence. J Appl Crystallogr 27:892–900. 23. Nardelli M. 1999. Modeling hydroxyl and water H atoms. J Appl Crystallogr 32:563–571. 24. Macrae, CF, Bruno IJ, Chisholm JA, Edgington PR, McCabe P, Pidcock E, Rodriguez-Monge L, Taylor R, van de Streek J, Wood PA. 2008. Mercury CSD 2.0 new features for the visualization and investigation of crystal structures. J Appl Crystallogr 41:466–470. 25. Sharma D, Gupta VK, Brahmachari G, Mondal S, Gangopadhyay A. 2007. X-ray study of weak interactions in two flavonoids. Bull Mater Sci 30:469–475.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
SpLine Spanish CRG Beamline at the ESRF. 6, Rue Jules Horowitz, BP 220, 38043 Grenoble Cedex 09, France
2
Instituto de Ciencia de Materiales de Madrid-ICMM/CSIC, Cantoblanco Madrid 28049, Spain
3
Departamento de Qu´ımica, Universidad de La Laguna. Instituto Universitario de Bio-Org´anica “Antonio Gonz´alez”, 38206 La Laguna, Tenerife, Spain
4 ´ de Rayos X, Universidad de La Laguna, 38206 La Laguna, Departamento de F´ısica Fundamental II, Servicio de Difraccion Tenerife, Spain
Received 14 June 2010; revised 24 September 2010; accepted 24 September 2010 Published online 24 November 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22379 ABSTRACT: The crystal structure of monohydrated trifolin (kaempferol 3-O-$-Dgalactopyranoside) (an important biologically active compound, which was isolated from the aerial part of Consolida oliveriana) has been determined from conventional laboratory X-ray powder diffraction data. Variable counting time technique was used during measurement and crystal structure was solved by means of Monte Carlo algorithm. The final structure was achieved by Rietveld refinement using both constraints and restraints on interatomic bond lengths and angles. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1588–1593, 2011 Keywords: X-ray powder diffractometry; crystal structure; crystallography; ab initio calculations; Monte Carlo
INTRODUCTION The naturally originated compounds belonging to the group of flavonoids, which are widely distributed in the human diet,1 have generated particular interest with regard to human health effects, including antioxidant activities2,3 or protection of cardiovascular diseases,4–6 and they have been a subject of intensive pharmacological studies in recent years, as they are among the most promising anticancer agents.7–9 In this context, some flavonoid complexes have been previously investigated10 (kaempferol, quercetin, trifolin, hyperoside, 2 -acetylhyperoside, 6 -acetylhyperoside, 7glucotrifolin, biorobin, or robinin). Monohydrated trifolin (C21 H20 O11 ·H2 O) is a derivative of flavonoid and, recently, it has been shown that the acetyl derivate presents cytotoxic properties.11 No suitable crystals ´ da Silva (Tel: +33-476-88-2449; Fax: Correspondence to: Ivan +33-476-88-2816; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 100, 1588–1593 (2011) © 2010 Wiley-Liss, Inc. and the American Pharmacists Association
1588
for single-crystal diffraction were obtained, but the structural information could be determined from powder diffraction data. Here, we report the ab initio structural determination of a monohydrated trifolin, using a Monte Carlo/ parallel tempering method, in order to obtain a starting model, followed by Rietveld refinement using constraints and restraints on bond lengths and angles.
EXPERIMENTAL Monohydrated trifolin was isolated from Consolida oliveriana. Details of extraction and isolation for the compound have been published previously.10 The sample was microcrystalline and attempts to grow single crystals were unsuccessful. Spectroscopic Study The structural identity of the studied compound (see, Fig. 1) was determined spectroscopically (proton magnetic resonance and 13 C nuclear magnetic resonance, infrared and ultraviolet-visible spectroscopy,
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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program13 ; the first 25 peaks, up to 29◦ in 22, were selected. Pattern indexing was performed with DicVOL06 program14 and a solution was obtained, which yielded a monoclinic cell with figures of merit15,16 of M(25) = 34.1, F(25) = 86.2 (0.0063, 46). For space group determination, Expo2004 program17 was used, where a statistical algorithm to determine the most probable space group have been implemented.18 In this case, the most probable extinction group was P 1 21 1, with a probability factor of 0.776. There are two compatible space groups (P21 and P21 /m) for this extinction group, but taking into account the cell and molecule volumes, they allowed us to choose only one (P21 ). Structure Determination by Monte Carlo Methods
Figure 1. Molecular structure of trifolin.
and mass spectrometry). The details of this study can be found elsewhere.10 X-Ray Diffraction The powder diffraction data were collected on a PANalytical X’Pert Pro diffractometer (PANalytical, Almelo, Netherlands) using Cu-K"1 radiation with a hybrid monochromator for parallel beam (Debye– Scherrer geometry-transmission mode). The sample was introduced in a Hilgenberg glass capillary (diameter = 0.3 mm) and spinning rotation was active to reduce the effect of possible preferential orientations. The detector was a X’Celerator (PANalytical, Almelo. Netherlands), working in continuous mode with 2.149◦ active length; soller slits of 0.02 radians were placed into the incident and diffracted beam path to reduce the asymmetry of peaks due to axial divergence. The 22 range was 3.5◦ –83◦ , with a step width of 0.0169◦ , and the total time of measurement was about 19 h. Variable counting time (VCT) procedure12 was used during data collection; it means that measurement time is increased toward higher 22 angles and it allows to get substantial amount of peaks information present also at high 22 angles, which is otherwise lost in conventional (i.e., fixed counting time) measurements. It is known that VCT procedure, in general, improves results on structure determination and more stable refinement of atoms coordinates and thermal parameters, especially for the organic compounds, are achieved.
RESULTS Data Reduction and Indexing Reflection positions were determined using the peak search algorithm implemented in the WinPLOTR DOI 10.1002/jps
Structure solution, by means of Direct Methods, was attempted with Expo2004 program, within P21 space group, without success. Thus, to obtain a starting structural model, Monte Carlo calculations were performed, using the parallel tempering algorithm implemented in the program FOX.19 A template of the trifolin molecule was built with the software package ChemBio Office (version 11.0), which was introduced in FOX, as well as a water oxygen. During the calculations, the observed and calculated intensities were compared only in the 22 range of 4◦ –52◦ and the molecule could translate and rotate randomly; torsion angles between the benzopyran and phenyl rings and between both benzopyran and glucose moiety and the oxygen atom connecting the two groups could also change, as well as the torsion angle C-OH in the glucose moiety. After ca. 13 million trials, the agreement factors were Rwp = 0.0694 and GoF = 10.054. Rietveld Refinement The solution found by FOX program was introduced in FullProf program20 to perform a Rietveld refinement. Atomic coordinates of the 33 independent nonH atoms were fitted, but constraints on benzopyran and phenyl rings and restraints on the other bond lengths and angles were introduced to limit the number of free parameters and ensure the convergence of the refinement process. The values for these bond lengths and angles were taken from similar molecules in The Cambridge Crystallographic Data Centre (CCDC) database and the mean-square deviations of assigned values were 0.01Å and 1◦ , respectively. Three different isotropic temperature factors were introduced: one for the water oxygen, other for the OH oxygen, and one for the rest of atoms. Intensities were corrected for absorption effects for a cylindrical sample. The peak function used was the Thompson— Cox–Hastings pseudo-Voigt,21 which can take into account the experimental resolution and the broadening JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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Figure 2. Comparison between the observed (red circles) and calculated (black continuous line, upper part) patterns of C21 H20 O11 ·H2 O. The difference curve (blue continuous line, lower part) and the reflection positions (green vertical lines) are also represented in the lower part of the figure.
due to size and strain effects, typical in this type of organic powder samples. The Finger’s treatment of the axial divergence22 was taken into account to model Table 1. Crystal Data and Structure Refinement for C21H20O11·H2O. Crystal data Formula Formula weight Cell setting, space group Temperature (K) a, b, c (Å); $ (◦ ) Volume (Å3 ) Z, Dc (g·cm−3 ) Radiation type : (mm−1 ) Specimen form, color Refinement Refinement method RF , RBRAGG Goodness-of-fit Wavelength (Å) Profile function No. of profile data steps No. of contributing reflections No. of bond length restraints No. of bond angle restraints
C21 H20 O11 ·H2 O 466.39 Monoclinic, P21 298 15.6461(3), 13.7636(2), 4.64731(8); 97.653(2) 991.87(3) 2, 1.56155 Cu K"1 1.010 Cylinder, white Rietveld refinement 0.0525, 0.0331 1.855 1.54056 Thompson–Cox–Hasting pseudo-Voigt 4671 693 25 41
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the asymmetry of the peak profile, and 69 points were chosen regularly distributed on the experimental pattern, in the 22 range 4–83◦ , to model the background through a linear interpolation made between two successive points. Hydrogen atoms for trifolin molecule were introduced in FullProf at their calculated positions with CALC-OH23 program, which combines geometric and force-field calculations on the basis of hydrogenbonding interactions, whereas H atoms’ positions for the water molecule were obtained with CCDC Mercury program.24 On the final Rietveld fit, there are 85 adjustable parameters (scale factor, zero shift, atomic coordinates, temperature factors, cell parameters, and peak shape), taking into account the introduced constraints. The final Rietveld agreement factors were Rp = 0.036, Rwp = 0.047, and χ2 = 3.44. In Figure 2, the plot of the final fit is given. Crystallographic and refinement-related data are reported in Table 1, whereas atomic coordinates and displacement parameters are reported in Table 2.
DISCUSSION The Oak Ridge Thermal Ellipsoid Plot Program drawing of the molecule monohydrated trifolin is shown in DOI 10.1002/jps
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Table 2. Atomic Coordinates and Isotropic Displacement Parameters (Å2) Obtained from Rietveld Refinement. Atom O1w O1 O6 O7 C1’’ C2 C2’’ C3 C3’’ C4 C4’’ C5’’ C6’’ H1’’ H2’’ H3’’ H4’’ H5’’ O2 O3 O4 O5 O8 O9 O10 O11 H3O H4O H5O H8O H9O H10O H11O H1OWa H1OWb H6’’a H6’’b C5 C6 C7 C8 C9 C10 H6 H8 C1’ C2’ C3’ C4’ C5’ C6’ H2’ H3’ H5’ H6’
x
y
z
Uiso
0.2232(10) 0.4065(6) 0.2577(8) 0.1151(5) 0.1888(5) 0.3267(8) 0.1661(6) 0.3220(12) 0.0975(6) 0.3778(10) 0.0184(8) 0.0436(5) −0.0310(9) 0.1987(5) 0.1493(6) 0.1034(6) −0.0334(8) 0.0536(5) 0.36275 0.4902(11) 0.6759(7) 0.1726(10) 0.2433(8) 0.0748(10) 0.0013(11) −0.0155(12) 0.4465(11) 0.6995(7) 0.1277(10) 0.2368(8) 0.1121(10) −0.0392(11) −0.0089(12) 0.246(13) 0.178(10) −0.0432(9) −0.0878(9) 0.51080 0.58150 0.59659 0.54099 0.47009 0.45490 0.61930 0.55099 0.29438 0.21220 0.16708 0.20349 0.28529 0.33109 0.18731 0.11210 0.30989 0.38579
0.83729 0.2176(11) 0.4151(13) 0.3993(10) 0.4489(6) 0.2665(8) 0.5552(6) 0.3605(7) 0.6016(6) 0.4106(8) 0.5455(6) 0.4384(6) 0.3715(6) 0.4376(6) 0.5611(6) 0.6007(6) 0.5588(6) 0.4315(6) 0.49713 0.4716(9) 0.1932(12) 0.0154(12) 0.6052(13) 0.6956(8) 0.5418(14) 0.2729(7) 0.4931(9) 0.2328(12) 0.0374(12) 0.6503(13) 0.7331(8) 0.5042(14) 0.2721(7) 0.819(15) 0.791(12) 0.3726(6) 0.3967(6) 0.38080 0.32771 0.23601 0.19892 0.25241 0.34350 0.35340 0.13752 0.20570 0.22980 0.16790 0.08061 0.05601 0.11731 0.28799 0.18480 −0.00218 0.09991
0.389(3) 0.311(3) 0.147(2) 0.163(3) 0.287(3) 0.243(3) 0.201(3) 0.315(4) 0.366(3) 0.572(4) 0.225(2) 0.279(3) 0.161(4) 0.497(3) −0.009(3) 0.578(3) 0.316(2) 0.491(3) 0.62870 1.045(3) 1.036(4) −0.663(3) 0.292(4) 0.256(4) −0.0800(19) 0.251(4) 0.950(3) 1.152(4) −0.750(3) 0.403(4) 0.331(4) −0.1196(19) 0.429(4) 0.21(3) 0.43(5) −0.074(4) 0.249(4) 0.92260 1.04060 0.93300 0.70469 0.58069 0.69119 1.19301 0.63278 −0.03210 −0.16251 −0.36252 −0.43372 −0.30471 −0.10300 −0.11510 −0.44972 −0.35361 −0.01520
0.092(9) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.061(2) 0.061(2) 0.061(2) 0.061(2) 0.061(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.078(2) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.116(4) 0.138(12) 0.138(12) 0.061(2) 0.061(2) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.061(2) 0.061(2) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.041(1) 0.061(2) 0.061(2) 0.061(2) 0.061(2)
Figure 3. The trifolin molecule consists of a benzopyran moiety, almost coplanar [0.71(15)◦ as dihedral angle between planes of benzene and pyran rings], a planar phenyl ring rotated by 10.68(13)◦ from the plane of the benzopyran ring system and a galactopyDOI 10.1002/jps
Figure 3. Oak Ridge Thermal Ellipsoid Plot Program drawing of the molecule trifolin in the asymmetric unit with labeling scheme
Figure 4. Molecular packing viewed along c axis.
ranoside ring adopting the 4C1 chair conformation with the benzopyran moiety positioned equatorially as substituent at C1’’. Bond distances and angles are comparable with values reported for similar flavonoids.25 The lengthening of the double bond C4 =O2 [1.238(12) Å] is due to strong intramolecular hydrogen bond between O2 and O3 . JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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Figure 5. Molecular stacking, showing the linking of the molecules by C−H · · · O and B–B interactions.
The hydroxyl group, O3 , has a gauche arrangement with respect to H3 -O3 -C5 -C10 torsion angle (1.74◦ ), giving rise to a short (1.831 Å) intramolecular contact between the H atom of the hydroxyl group and carbonyl atom O2 . The projection of the crystal structure down the c axis is shown in Figure 4. Weak intermolecular interactions play a decisive role in determining the three-dimensional structure for this compound, with the presence of short contacts of C−H · · · O type and B–B interactions with a centroid–centroid distance of 4.761 Å (see, Fig. 5). Final position and orientation of the water molecule allows us to conclude that the water molecule plays a stabilizing role in the molecular packing (see, Figs. 4 and 5).
CONCLUSION In this study, we have shown the structure determination of monohydrated trifolin compound from conventional laboratory X-ray powder diffraction data, using a VCT procedure during measurement. This allowed us to determine the structure by means of Monte Carlo methods and refine the model using the Rietveld method. Recent advances on algorithms aiming to solve crystal structures using powder diffraction data allow us to deal, in an easier way, new structural studies on compounds in which only powder samples can be obtained, as is the case of monohydrated trifolin. CCDC 775168 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data request/cif, or by emailing data request@ccdc. cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 4, APRIL 2011
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