551 Research Article Received: 17 September 2008 Revised: 4 February 2009 Accepted: 8 February 2009 Published online in Wiley Interscience: 16 April 2009 (www.interscience.com) DOI 10.1002/mrc.2427 1 H NMR discrimination of CHF4226.01 diastereoisomers in DMSO-d 6 Silvia Capacchi, * a Milco Lipreri, a Andrea Rizzi, b Claudia Cal ´ o, b Tiziana Peveri a and Silvia Catinella a Two diastereoisomers CHF4226.01 (R, R) and CHF4232.01 (S, R), differing for a chiral center, have been studied to investigate their possible discrimination using NMR. 1D NMR and 2D NMR experiments, such as COSY, NOESY and ROESY, were performed on pure isomers and on the association complexes formed in the presence of the chiral reagent (S)-(-)-1-(2-napthyl)ethylamine (S-NEA). Moreover, computational studies, concerning conformational analysis and molecular dynamics, were started and supported the NMR results. Copyright c  2009 John Wiley & Sons, Ltd. Keywords: NMR; 1D; 2D NMR; COPD; diastereoisomers; chiral reagents; association complexes; molecular modeling Introduction Carmoterol, 8-hydroxy-5-[(1R)-1-hydroxy-2-[N-((1R)-2-(4- methoxyphenyl)-1-methylethyl)amino]ethyl]-carbostyrilhydro- chloride β 2 agonist, is known as a bronchodilator for the relief of airflow limitation in lung diseases such as asthma and potentially in chronic obstructive pulmonary disease (COPD). In this field of research, both CHF4226.01 (R, R) and its diastereoisomer CHF4232.01 (S, R) (Fig. 1) were investigated by using NMR. In fact, normal and chiral HPLC analyses were performed onto both diastereoisomers but results were quite poor. Classical capillary electrophoresis was also used, but it is a time wasting technique, and our NMR analyses resulted much faster than capillary electrophoresis and not sample destroying. Therefore, we developed a new NMR application of the chiral recognition method, which could easily be applied in every laboratory and that quickly became our new routine technique to discriminate between diastereoisomers. No crystal suitable for X-ray analysis was ever obtained for CHF4226.01. But its precursor CHF4190.01, benzylated on the OH group of the carbostyril moiety, was obtained as a single crystal and the X-ray determination confirmed the absolute configuration of its stereocenters (R, R). Actually, the deprotection leading from CHF4190.01 to CHF4226.01 does not affect the chiral centers. Therefore, to further try to investigate their structure, CHF4226.01 (R, R) and its diastereoisomer CHF4232.01(S, R) (Fig. 1) were studied by using NMR. Both one-dimensional and two-dimensional NMR sequences were used for an in-depth study of the two compounds. First of all, the one-dimensional 1 H NMR spectra of the pure diastereoisomers were performed to possibly point out any small difference between CHF4226.01 (R, R) and CHF4232.01 (S, R). Subsequently, the chiral reagent (S)-(−)-1-(2-napthyl)ethylamine (S-NEA) was added to each diastereoisomer, hopefully to form association complexes showing bigger shift effects than the isomers alone. In fact, it is very well known in literature that chiral reagents can enhance shielding/deshielding effects and help resolve diastereoisomers and/or enantiomers. [1 – 11] In parallel, 2D NMR experiments such as COSY, NOESY and ROESY were run on diastereoisomers alone CHF 4226 (R,R) CHF 4232 (S,R) N H O OH HN CH 3 HO O CH 3 HCl R S N H O OH HN CH 3 HO O CH 3 H 5 H 4 ' H 7 H 3 H 9 R R HCl 10 14 13 11 12 4 4 6 6 1 1 2 8 Figure 1. Diastereoisomers. and in the presence of S-NEA to provide and confirm 1D NMR assignments. Experimental Materials Diastereoisomers CHF4226.01 (R, R) and CHF4232.01 (S, R) were synthesized in Chiesi at the Chemical Synthesis Department. The chiral reagent (S-NEA) was from Fluka. Solvent DMSO-d 6 , 99.96% pure, was from Euriso-Top. Preparation of the NMR samples Pure diastereoisomers CHF4226.01 and CHF4232.01 Working in an atmosbag saturated with argon, CHF4226.01 (or CHF4232.01) (8.1 mg, 0.02 mmol) was dissolved into 0.75 ml ∗ Correspondence to: Silvia Capacchi, Chiesi Farmaceutici s.p.a, Analytical Chemistry Department, Via S. Leonardo 96, 43100 Parma, Italy. E-mail: S.Capacchi@chiesigroup.com a Chiesi Farmaceutici s.p.a, Analytical Chemistry Department, Via S. Leonardo 96, 43100 Parma, Italy b Chiesi Farmaceutici s.p.a, Computational Chemistry Unit, Chemical Synthesis Department, Via S. Leonardo 96, 43100 Parma, Italy Magn. Reson. Chem. 2009, 47, 551–561 Copyright c  2009 John Wiley & Sons, Ltd.