AD-A262 979 13C NMR SPECTRA OF ALLOSTERIC EFFECTORS OF HEMOGLOBIN Stanislaw Ostrowskil) Thomas G. Burke, and Waldemar Priebe The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA [SO, WPI and College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA [TGBI KEY WORDS: Allosteric effectors of hemoglobin, Urea and thiourea derivatives, 13C NMR, Spectral assignments. INTRODUCTION In the last three years many papers dealing with the preparation and biological activity of compounds that act as allosteric effectors of hemoglobin have been published.2-4) Among the numerous moieties, some urea derivatives were found to be very effective in reducing oxygen affinity of hemoglobin.2,3) The structures of most of these compounds were confirmed by IH NMR spectra; however, to our knowledge, 13C NMR spectra have not been published for any of these allosteric modifiers of hemoglobin. We prepared in this series over 30 new urea and thiourea derivatives with the intent of investigating their biological activity.s) An analysis of 13C NMR spectra revealed high regularity in the chemical shifts of the similar fragments of the structures and revealed signal deviations among structures having different substituents in the aromatic rings. RESULTS AND DISCUSSION A simple analysis procedure was used to assign peaks in the spectra. At first, two peaks corresponding to the signals of the aromatic rings A and B were 2 493-07620 identified. For R = H in all cases the chemical shifts originating from C-4, C-5(5'), C- 6(60, and C-7 were very close or correspondingly identical (e.g., 18 and 20, Table 1), whereas the C(9) - C(14) signals varied, depending on substituents in ring A. A replacement of R - H by the C(CH )2CO H group is reflected in the 8 values of the 3 2 C(4) -C(7) signals, and virtually did not change the C(9) -C(14) signals in the respective pairs of compounds (e.g., 13, 14). For this series ( R - C(CH ) CO H, R1, R2 - 3 2 2 changeable) the very close occurrence of the corresponding C(4) - C(7) chemical shifts was observed as well (e.g., 14 and 28). On this basis the bulk of the signals (spectra recorded with C-H decoupling technique) was assigned to the appropriate carbons ir; both aromatic rings. Independently, the calculations of 8 values from increments6) for all aromatic carbon atoms were done. We found good agreement between the chemical shifts calculated from increments and those experimentally determined (e.g., compound 1, calculated: C-4 (149.8 ppm), C-5,5' (116.0), C-6,6' (120.0), C-7(132 7), C-9 (141.0), C-10 (133.9), C-11 (130.1), C-12 (117.6), C-13 (145.2), C-14 (113.9), C-11 (130.1), C-12 (117.6), C-13 (145.2), C-14 (113.3); compound 1, experimental data: 152.8, 115.3, 120.4, 131.2, 138.9, 134.0, 130.7, 116.2, 146.1, 113.3 ppm respectively). For some instances a coupling between carbon atoms and fluorine was diagnostic and helpful for correct assignment. In 15 we were left with two unidentified peaks at 106.2 (broader singlet) and 105.7 (d, J = 5 Hz), originating from C-10 and C-14. Singlet 106.2 was assigned to C-10 owing to the absence of meta carbon-fluorine coupling. This lack of a three-bond coupling constant was revealed when any substituent was present in aromatic ring between the respective centers (see 14-18). Ortho interactions deformating the planarity of arene ring (together with substituents) also decreased 2JCF values, especially when the neighboring group (to fluorine) was relatively bulky, e.g., constants of C-8 and C-10 carbons with fluorine in 14. In some cases a long-range influence of the substituents on the chemical shifts was observed. When R = H was replaced by the C(CH )2CO H group, not only 3 2 the (C-4) and (C-5,5') were changed, but also (C-7) was shifted downfield by c.a. 3-4 ppm. This change was relatively smaller on (C-6,6'), c.a. 1.5 ppm. In this series the C- 5(5'), C-6(6') carbons were readily identified by their high intensity, but it was difficult to correctly assign them because of a close appearance of their chemical shifts. For two selected compounds (6, 12) two-dimensional measurements were recorded. In these cases downfield shifted signals corresponded with C-5(5') protons. On this basis we propose to assign (C-5, 5') and (C-6, 6') values for the remaining compounds in this series. An introduction of an NH group into terminal aromatic ring A causes not 2 only dramatic changes in the neighboring carbon atom chemical shifts. Also were observed, independently which position NH group occupies (compounds: 5, 6, 9, 2 12, 15), long-range effects on C-8 (small downfield shift), C-7 (downfield shift) and even C-4 (upfield shift -1 ppm). The neighboring amino group also changed the carbon-fluorine coupling, decreasing it up to 25 Hz (compound 15). Significant changes in chemical shifts were caused by a thiocarbonyl group in the urea bridge. Carbon atoms C-6(6'), C-1C, and C-14, situated in the same distance from C=S are shifted downfield by over 5 ppm in 22, 23, 26 as compared with corresponding urea analogues 20, 21, 25. Interestingly, that even five-bond distanced from C=S C-4 and C-12 are moved downfield by over 2 ppm. this influence is relatively smaller on C-7 and C-9. EXPERIMENTAL All 13C and two-dimensional NMR measurements were carried out in 5 mm tubes in DMSO-d using a QE-300 spectrometer operating at 75.234 MI-Iz for 13C and a 6 Bruker AM-400 spectrometer operating at 100.623 MHz for 13C and at 400.139 MHz for 1H. Sample concentrations were approximately 30 mg mi-r. Other experimental data for 13C NMIR were: pulse width, 4.0 gs; acquisition time, 0.27 s; flip angle, 90'; and spectral width 20 kHz. Corresponding data for 1H were: pulse width, 8.6 As; acquisition time, 0.8 s; flip angle, 10'; and spectral width, 5 kHz. All data were referenced to DMSO-d at 39.50 ppm. Synthesis and chemical characterization of the 6 compounds 1-30 and 32-33 will be published elsewhere.5). Data for other new compounds were as follows (melting points were uncorrected): 1-(3-Trifluoromethylphenyl)-3-(4-hydroxyphenyl) urea (31). Compound 31 was prepared according a procedure described in the literature3) and resulted in 90% yield, m.p. 202"C (CHCI /MeOH). For C 4I-InF N 0 (296.25): 3 1 3 2 2 Calcd C 56.76, H 3.74, N 9.46. Found C 56.66, H 3.75, N 9.50. 1-(4-Methylthiophenyl)-3-(4-hydroxyphenyl) urea (34). Compound 34 was prepared as above, resulting in 96% yield, m.p. 214"C (CHCI /McOH). For C14H1 N 0 S 3 4 2 2 (274.34): Calcd C 61.29, H 5.14, N 10.21, S 11.69. Found C 61.08, H 5.19, N 10.18, S 11.79. ACKNOWLEDGMENTS This work was supported by Office of Naval Research Grant No. N00014-90-j- 1648. References 1. Current address: Institute of Organic Chemistry, Polish Academy of Sciences, 01-224, Warsaw, Poland. 2. I. Lalezari, S. Rahbar, P. Lalezari, G. Fermi, M. F. Perutz, Proc. Natl. Acad. Sci. U.S.A., 85, 6117 (1988). 3. I. Lalezari, P. Lalezari, J. Med. Chem., 32, 2352 (1989). xLo (cid:127) . "5! 5 4. R.S. Randad, M. A. Mahran, A. S. Mehanna, D.J. Abraham, J. Med. ((9q1& Chem., 34,752 (1991); F.C. Wireko, G.E. Kellogg, D.J. Abraham, ibid., 34, 758 (1991); D.J. Abraham, F.C. Wireko, R.S. Randad, C. Poyart, J. Kister, B. Bohn, J.-F. Liard, and M.P. Kunert, Biochem.,31, 9141 (1992). 5. T.G. Burke, S. Ostrowski, S. Rahbar, and W. Priebe. Pharmaceut. Res. 9: S-74 (1992). 6. P. Clerc, S. Simon, Tables of Spectral Data for Structure Determination of Organic Compounds, edited by F.L. Boschke et al., Springer-Verlag, Berlin, Heildelberg, New York, Tokyo, 1983, p. 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