1 H, 13 C NMR studies of new 3-aminophenol isomers linked to pyridinium salts Santiago Schiaffino-Ortega, Antonio Espinosa, Miguel A. Gallo, L. Carlota López-Cara* and Antonio Entrena* 1 H and 13 C NMR spectroscopic data of 20 new non-symmetrical compounds were assigned by a combination of 1D and 2D NMR experiments (DEPT, HSQC, and HMBC). These compounds contain a 4-(N,N-dimethylamino)- or 4-(pyrrolidin-1-yl)pyridinium moiety and a 3-nitro-, 3-amino-, or 3-hydroxyphenyl ring, linked by p-xylene, 4,4′-dimethylbiphenyl, 1,2-bis(p-tolyl)ethane, or 1,4-bis(p-tolyl)butane. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: 1 H– 13 C 1D NMR; 1 H– 13 C 2D NMR (HSQC; HMBC); choline kinase inhibitors; antiproliferative compounds Introduction Evidence for the requirement of choline kinase-α activity in cancer has been obtained from observations that the expression of this enzyme is really high in many tumor cells. [1] Theoretical studies initially performed on a human choline kinase homology model [2] and further on the X-ray crystal struc- ture of the enzyme indicated that non-symmetrical inhibitors could insert the cationic head into the choline binding site and the adenine moiety into the ATP binding site. [3] In a recent paper, the synthesis and the biological evaluation of a novel family of compounds bearing a 3-aminophenol fragment and a pyridinium salt connected by means of different linkers, which were designed and evaluated as ChoK inhibitors and as antiproliferative agents, have been described. [4] Although the structures of these derivatives have been determined by means of standard spectroscopic techniques ( 1 H and 13 C NMR, and MS), a detailed NMR study has been performed in some of them, in order to unequivocally corroborate their structures. This paper reports the 1 H and 13 C NMR unambiguous signal assignments corresponding to the pyridinium salts, the linkers, and the 3-aminophenol moiety (4a–h and 5a–h) using 1D and 2D NMR techniques. The spectra of nitro derivatives 3e–h, precursors in the synthetic pathway for the preparation of compounds 4a–h, are also included. Experimental NMR spectra were recorded on 500 MHz 1 H and 125 MHz 13 C NMR Agilent Direct-Drive (Santa Clara, CA, USA), 400 MHz 1 H NMR Agilent Direct-Drive, or 300 MHz 1 H and 75 MHz 13 C NMR Agilent Inova-Unity spectrometers at 298 K. Chemical shifts (δ) are quoted in parts per million (ppm) and are referenced to the residual solvent peak: CD 3 OD, δ = 3.31 ppm ( 1 H), δ = 49.05 ppm ( 13 C); DMSO-d 6 , δ = 2.50 ppm ( 1 H), δ = 39.5 ppm ( 13 C). Spin multiplicities are given as s (singlet), bs (broad singlet), d (doublet), dd (doublet doublet), ddd (doublet, doublet doublet), t (triplet), dt (doublet triplet), q (quadruplet), and m (multiplet). Coupling constants (J) are given in Hz. The following parameters were used in DEPT experiments: PW (Pulse Width: 135°), 9.0 ms; recycle time, 1 s; 1/2 J (CH) = 4 ms; 65.536 data points acquired and transformed from 1024 scans; spectral width, 15KHz; and line broadening, 1.3 Hz. HMBC spectra were measured with a pulse sequence gc2hmbc (Standard sequence, Agilent Vnmrj_3.2A software) optimized for 8 Hz (inter-pulse delay for the evolution of long- range couplings: 62.5 ms). The HSQC spectra were measured with a pulse sequence gc2hsqcse (Standard sequence Agilent Vnmrj_3.2A software). Results and Discussion Scheme 1 represents the previously reported synthetic pathway followed in the preparation of two families of compounds 4a–h and 5a–h. [4] Compounds with general structure 2 were prepared using linkers 1a–d, which were synthetized following the reported route. [4,5] Reaction of compounds 1a–d with a half equivalent of 4-(N,N-dimethylamino)pyridine or 4-pyrrolidinopyridine, in butanone during 3 days at room temperature, yields the corre- sponding derivatives 2a–h. Compounds 3e–h were obtained by treatment of compounds 2a–h with 3-nitrophenol in the presence K 2 CO 3 , with general good yields. Preparation of compounds 5a–h needs an initial protection of the OH group in 3-aminophenol and a further reaction with intermediates 2a–h in the presence of K 2 CO 3 , being the OH group liberated during this last reaction. Finally, compounds 4a–h were obtained using two routes depending on the starting intermediate. In this sense, compounds 5a–d were obtained by reaction of 3-aminophenol and intermediates 2a–d in the presence of NaH, while reduction * Correspondence to: L. C. López-Cara and Antonio Entrena, Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071 Granada, Spain. E-mail: lcarlotalopez@ ugr.es; aentrena@ugr.es Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071, Granada, Spain Magn. Reson. Chem. (2013) Copyright © 2013 John Wiley & Sons, Ltd. MRC letters Received: 3 September 2013 Revised: 30 September 2013 Accepted: 5 October 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/mrc.4025