620 Chem. Pharm. Bull. 66, 620–623 (2018) Vol. 66, No. 6 Regular Article Chiral Recognition of Pharmaceuticals Having a Xanthine Skeleton by ()-Epigallocatechin-3-O-gallate in Water Hiroyuki Tsutsumi, Haruka Tanabe, and Takashi Ishizu* Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University; Sanzo Gakuen-cho 1, Fukuyama, Hiroshima 729–0292, Japan. Received January 16, 2018; accepted March 5, 2018 A mixture of pharmaceuticals having a xanthine skeleton, theophylline, proxyphylline, diprophylline and ()-epigallocatechin-3-O-gallate (EGCg) in water created a sticky precipitates, which were thought to be 2 : 2 complexes of the pharmaceuticals and EGCg. The molecular capture ability of the pharmaceuticals having a xanthine skeleton by EGCg was estimated by the amount of the pharmaceuticals included in the precipitates of the complexes, and measured by the integrated value of proton signals in the quantitative 1H-NMR spectra. Based on changes in chemical shifts of proton signals of the pharmaceuticals with a xan- thine skeleton in 1H-NMR spectra by adding standard amounts of EGCg, the xanthine skeleton of the phar- maceuticals was considered to exist in the hydrophobic space formed by the three aromatic A, B, B rings of EGCg, and a part of the proxyphylline and diprophylline side chains existed out of the hydrophobic space. In the 1H-NMR spectra of the mixture of (R)- and (S)-proxyphylline, (R)- and (S)-diprophylline and an equi- molecular amount of EGCg, the N3-CH3 signal of (R)- and (S)-proxyphylline, and (R)- and (S)-diprophylline was clearly observed as two singlets. This suggested that EGCg recognized the chirality of proxyphylline and diprophylline in water. Key words chiral recognition; (−)-epigallocatechin-3-O-gallate; 1H-NMR; pharmaceutical having xanthine skeleton; proxyphylline; diprophylline Catechins are a group of polyphenols that occur naturally in Pro residue.7) Based on the crystal structures of the 2 : 2 com- certain species of plants, including tea (Camellia sinensis, Ca- plexes of EGCg and cyclo(L-Pro-Gly), cyclo(D-Pro-Gly), such melliaceae), and are major ingredients in green tea infusions. a difference in chemical shift may have been due to magnetic The physiological activity and function of catechins have been anisotropic shielding effects by the ring current from the B studied by many researchers. The role of such molecules in the ring of EGCg (Fig. 1). prevention of cancer and cardiovascular disease has received However, the splitting of the methylene protons of the Pro a great deal of attention.1,2) Catechins may be scavengers of residue of cyclo(Pro-Gly) was difficult to observe in the chiral reactive oxygen species, which are thought to cause several recognition due to small differences in their chemical shifts diseases such as cancer, lifestyle diseases, aging, etc.3,4) and overlap of signals.7) In this report we applied chiral recog- We previously studied the molecular recognition, molecu- nition to pharmaceuticals, which are currently used in a race- lar capture, and asymmetric recognition of tea catechins in mic mixture. Thus, the chiral recognition of pharmaceuticals water. The gallated catechin (−)-epigallocatechin-3-O-gallate with a xanthine skeleton for bronchial asthma, proxyphylline (EGCg), which is most abundant in tea catechins, formed a and diprophylline were investigated using EGCg in water (Fig. hydrophobic space surrounding the top and lower walls of 2). As pharmaceuticals having a xanthine skeleton have two the B′ rings of EGCg, and right and left walls of the A and B methyl groups, which were observed as a sharp, strong singlet, rings of EGCg in water. Caffeine5) and nicotineamide,6) which the split of the methyl signal was considered as easy to ob- are also components of tea, were captured by the hydrophobic serve in 1H-NMR spectra. space formed by the three aromatic A, B, B′ rings of EGCg, and 2 : 2 complexes of EGCg were formed. As a result, the Experimental 2 : 2 complex precipitated from the aqueous solution due to its NMR Experiments 1H-NMR spectra were recorded with hydrophobicity. a JEOL JMN-LA500 (Tokyo, Japan) operating at 500 MHz. Furthermore, it was assumed that the space formed by the D2O (99.9 atom% D; Wako Pure Chemical Industries, Ltd.) three aromatic A, B, B′ rings of EGCg recognized the chiral- was used as a solvent. Chemical shift values are expressed ity of compounds included in the space because the C ring of in ppm downfield using sodium 2,2-dimethyl-2-silapentane- EGCg has two chiral carbon atoms, C2 and C3, and the hydro- 5-sulfonate (DSS) as an internal standard. The nuclear Over- phobic space formed by the three aromatic A, B, B′ rings of hauser effect (NOE) difference experiments were typically EGCg was a chiral space. conducted with 32 K data points covering a spectral width of On this assumption, we attempted the chiral recognition 10000 Hz and with ca. 5s presaturation time. 1 of diketopiperazines cyclo(L-Pro-Gly) and cyclo(D-Pro-Gly) Quantitative H-NMR (qNMR) Experiments qNMR by EGCg. Upon formation of 2 : 2 complexes of EGCg and was performed with the following optimized parameters: cyclo(L-Pro-Gly), cyclo(D-Pro-Gly) in D2O, the chirality of probe temperature, 25°C; spinning, off; number of scans, cyclo(Pro-Gly) was recognized by a difference in the chemi- eight; spectral width, 20 ppm; relaxation delay, 64 s; pulse 1 cal shift of H-NMR signal for some methylene protons of the angle, 90°; internal standard, DSS-d6 (Wako Pure Chemical * To whom correspondence should be addressed. e-mail: [email protected] © 2018 The Pharmaceutical Society of Japan Vol. 66, No. 6 (2018) Chem. Pharm. Bull. 621 Fig. 1. Intermolecular Interactions of 2 : 2 Complexes of EGCg and Cyclo(Pro-Gly) (a) 2 : 2 complex of EGCg and cyclo(L-Pro-Gly). (b) 2 : 2 complex of EGCg and cyclo(D-Pro-Gly). Black arrows and dotted lines indicate CH–π interactions and hydrogen bonds, respectively. Table 1. The Molecular Capture Ability of EGCg for the Pharmaceuti- cals Having a Xanthine Skeleton Xanthine derivative A (%) B Pyridine 49.47 1.000 Theophylline 86.60 1.751 Proxyphylline 79.32 1.603 Diprophylline 90.15 1.822 A: Mole number of each hetelocyclic compound in a crude precipitate/total mole number. B: A relative ratio when pyridine is set to 1.000. Fig. 2. Proxyphylline and Diprophylline Industries, Ltd.). Dimethyl sulfoxide (DMSO)-d6 (99.9 atom% D; Wako Pure Chemical Industries, Ltd.) was used as a sol- vent and DSS-d6 (Wako Pure Chemical Industries, Ltd.) was used as an internal standard. Preparation and qNMR of Sticky Precipitate Formed by EGCg and Pharmaceuticals Having a Xanthine Skel- eton EGCg (1.09×10−2 mmol) and pharmaceuticals having −2 a xanthine skeleton (1.09×10 mmol) were dissolved in D2O (70 µL) at 90°C, and left at room temperature for a day to ob- tain a supernatant liquid and sticky precipitate. After remov- ing the supernatant liquid, the sticky precipitate was evapo- rated under reduced pressure to create a residue. The residue was dissolved in DMSO-d6 (520 µL) contain- −3 ing DSS-d6 (1.44×10 mmol). The content of the resulting DMSO-d6 solutions was measured by qNMR with the methyl Fig. 3. The Chemical Shift Change of the Theophylline Protons by Adding EGCg group of DSS-d6 as an internal standard. Initial condition: the solution of theophylline (10 mM) in D2O at at 35°C. Results and Discussion Complex Formation of EGCg with the Pharmaceuticals of EGCg, and the pharmaceuticals having xanthine were cap- Having a Xanthine Skeleton Equimolecular amounts of tured in the hydrophobic space from the aqueous solution. pharmaceuticals having a xanthine skeleton, theophylline, The molecular capture ability of the pharmaceuticals with proxyphylline, diprophylline and EGCg in an aqueous solu- a xanthine skeleton by EGCg was estimated by the amount of tion divided into a supernatant liquid and a sticky precipitate, pharmaceuticals included in the precipitates of the complexes, which contained the pharmaceuticals with a xanthine skeleton which was measured by the integrated value of proton signals and EGCg at a molar ratio of 1 : 1 based on measurement of in the 1H-NMR spectra (Table 1). the integral volume of 1H-NMR signals. The precipitates were The Pharmaceuticals Having a Xanthine Skeleton in thought to be 2 : 2 complexes of the pharmaceuticals with the EGCg Complex Changes in chemical shifts of proton a xanthine skeleton and EGCg such as the 2 : 2 complex of signals of theophylline in 1H-NMR spectra by adding standard cyclo(Pro-Gly) and EGCg7) (Fig. 1). The 2 : 2 complexes had amounts of EGCg were observed. Upfield shifts in proton a hydrophobic space formed by three aromatic A, B, B′ rings signals for all protons N1-CH3, N3-CH3 and H8 were observed 622 Chem. Pharm. Bull. Vol. 66, No. 6 (2018) (Fig. 3). xanthine skeleton and C10 in its side chain were included in It was thought that the upfield shifts of proton signals re- the hydrophobic space formed by the three aromatic A, B, B′ sulted from the magnetic anisotropic shielding by the ring rings of EGCg in the 2 : 2 complex, and C11 and C12 of the side current from the B′ rings of EGCg, and the whole theophyl- chain existed out of the hydrophobic space, as shown in Fig. line molecule was captured by the hydrophobic space formed 6b. by EGCg, such as the cyclo(Pro-Gly) moieties of the 2 : 2 com- Similarly, changes in chemical shifts of proton signals of plex of cyclo(Pro-Gly) and EGCg, as shown in Figs. 1 and 6a. (R)- and (S)-diprophyllines8) in 1H-NMR spectra by add- Next, changes in chemical shifts of proton signals of (R)- ing standard amounts of EGCg were observed (Figs.