D. Castagnolo et al. / Bioorg. Med. Chem. 16 (2008) 8587–8591
8591
5.4. Parallel synthesis of pyrazoles 9a–f
trose-catalase enrichment. Final inoculum was 1.5 Â 103 per well
and was obtained as described previously.14 Plates were incubated
for 21–28 days and MICs were read as minimal concentrations of
compounds completely inhibiting visible growth of mycobacteria.
Compounds 1 and 2 (0.5 mmol),4 divided into six different ves-
sels, were placed in the Büchi SyncoreÒ and dissolved in dry DMF
(5 mL). NaH (2 equiv/mol) was added and the reaction mixtures
were stirred at 300 rpm for 1 h. The appropriate benzyl chloride
(or benzyl bromide) (1 equiv/mol) and NaI (cat.) were then added
and the resulting mixtures were stirred at rt at 300 rpm overnight.
Water (5 mL) and AcOEt (5 mL) were added and the resulting mix-
tures were stirred for 1 h. The organic layers were then filtered out
in parallel and evaporated to dryness to afford crude compounds
9a–f, which were purified by flash chromatography (hexanes/ethyl
acetate 4:1) to afford the final products (20–73% yield).
7. Computational details
Computational analysis was performed by means of the Catalyst
software package, version 4.10.10
All the compounds were built using the 2D/3D sketcher of the
program. A representative family of conformations was generated
for each molecule using the CHARMm force field implemented in
Catalyst, together with the Poling algorithm and the best quality
conformational analysis method.15,16 Conformational diversity
was emphasized by selection of the conformers that fell within a
20 kcal/mol range above the lowest-energy conformation. The
Compare/Fit command in the Hypothesis Generation workbench
was used to analyze the mapping mode of compounds within the
pharmacophoric model. In particular, the Best Fit option was ap-
plied, which manipulates conformers within the specified energy
threshold to minimize the distances between hypothesis features
and mapped atoms in the molecule.
9a: Yield: 50%. 1H NMR (CDCl3): d (ppm) 7.73 (2H, d, J = 8.13 Hz,
Ph), 7.38–7.28 (5H, m, Ph), 6.90 (2H, d, J = 7.71 Hz, Ph), 6.83–6.81
(2H, m, Ph), 6.81–6.79 (2H, m, Ph), 4.82 (2H, s, NCH2Ph), 2.57
(3H, s, CH3). MS: m/z 421–423 (M+1)+; 443–445 (M+Na)+; 863
(2M+Na)+. Anal. for C24H18ClFN2O2 Calcd.%: C, 68.49; H, 4.31; N,
6.66. Found%: C, 68.63; H, 4.32; N, 6.67.
9b: Yield: 20%. 1H NMR (CDCl3): d (ppm) 7.76 (2H, d, J = 8.33 Hz,
Ph), 7.39–7.7.15 (10H, m, Ph), 6.88–6.86 (2H, m, Ph), 4.88 (2H, s,
NCH2Ph), 2.61 (3H, s, CH3). MS: m/z 403–405 (M+1)+; 425–427
(M+Na)+; 827–829 (2M+Na)+. Anal. for C24H19ClN2O2 Calcd%: C,
71.55; H, 4.75; N, 6.95. Found%: C, 71.76; H, 4.76; N, 6.97.
9c: Yield: 60%. 1H NMR (CDCl3): d (ppm) 8.09 (2H, d, J = 8.61 Hz,
Ph), 7.75 (2H, d, J = 8.52 Hz, Ph), 7.37–7.28 (5H, m, Ph), 7.13 (2H, d,
J = 7.7 Hz, Ph), 7.02 (2H, d, J = 8.61 Hz, Ph), 4.94 (2H, s, NCH2Ph),
2.58 (3H, s, CH3). MS: m/z 448–450 (M+1)+; 470–472 (M+Na)+;
486 (M+K)+; 917–919–918 (2M+Na)+. Anal. for C24H18ClN3O4
Calcd%: C, 64.36; H, 4.05; N, 9.38. Found%: C, 64.62; H, 4.06; N,
9.42.
Acknowledgments
Financial support from the Italian Ministero dell’Istruzione,
dell’Università e della Ricerca (PRIN 2005037820) is gratefully
acknowledged. CARIPLO (Grant Rif. 2006.0880/10.8485 Bando
2006) is gratefully acknowledged. We thank Asinex for partial
financial support for this work.
9d: Yield: 73%. 1H NMR (CDCl3): d (ppm) 7.75 (2H, d, J = 8.28 Hz,
Ph), 7.37–6.80 (10H, m, Ph), 4.82 (2H, s, NCH2Ph), 2.60 (3H, s, CH3).
MS: m/z 455–457 (M+1)+; 477–479 (M+Na)+; 933–931 (2M+Na)+.
Anal. for C24H17Cl2FN2O2 Calcd%: C, 63.31; H, 3.76; N, 6.15. Found%:
C, 63.76; H, 3.77; N, 6.16.
References and notes
1. (a) World Health Organization: The World Health Organization Global
2003, 83, 44; (c) Frieden, T. R.; Munsiff, S. S. Clin. Chest Med. 2005, 26, 197;
(d) Burman, W. J. Clin. Chest Med. 2005, 26, 283.
3. (a) Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479; (b) O’Brien, R. J.; Nunn, P. P.
Am. J. Respir. Crit. Care Med. 2001, 1635, 1055; (c) Ahmad, Z.; Pandey, R.;
Sharma, S.; Khuller, G. K. Int. J. Antimicrob. Agents 2008, 31, 142.
4. Manetti, F.; Magnani, M.; Castagnolo, D.; Passalacqua, L.; Botta, M.; Corelli, F.;
Saddi, M.; Deidda, D.; De Logu, A. ChemMedChem 2006, 1, 973.
5. Kutterer, K. M.; Davis, J. M.; Singh, G.; Yang, Y.; Hu, W.; Severin, A.; Rasmussen,
B. A.; Krishnamurthy, G.; Failli, A.; Katz, A. H. Bioorg. Med. Chem. Lett. 2005, 15,
2527.
9e: Yield: 20%. 1H NMR (CDCl3): d (ppm) 7.78 (2H, d, J = 8.00 Hz,
Ph), 7.41–7.13 (9H, m, Ph), 6.92–6.90 (2H, m, Ph), 4.91 (2H, s,
NCH2Ph), 2.66 (3H, s, CH3). MS: m/z 437–439 (M+1)+; 459–461
(M+Na)+; 895–897 (2M+Na)+. Anal. for C24H18Cl2N2O2 Calcd%: C,
65.91; H, 4.15; N, 6.41. Found%: C, 66.04; H, 4.16; N, 6.42.
9f: Yield: 30%. 1H NMR (CDCl3): d (ppm) 8.13 (2H, d, J = 8.75 Hz,
Ph), 7.77 (2H, d, J = 7.99 Hz, Ph), 7.40–7.31 (4H, m, Ph), 7.15–7.06
(4H, m, Ph), 4.97 (2H, s, NCH2Ph), 2.61 (3H, s, CH3). MS: m/z 504–
506 (M+Na)+; 987 (2M+Na)+. Anal. for C24H17Cl2N3O4 Calcd%: C,
59.77; H, 3.55; N, 8.71. Found%: C, 59.95; H, 3.56; N, 8.73.
6. Pyrazolones 7a–k can exist in three tautomeric forms as indicated in Scheme 1.
1H NMR analysis revealed the presence of a single signal set due to the quick
chemical exchange between the three tautomeric forms.
7. Jensen, B. S. Acta Chem. Scand. 1959, 13, 1668.
8. Holzer, W.; Kautsch, C.; Laggner, C.; Claramunt, R. M.; Pérez-Torralba, M.;
Alkorta, I.; Elguero, J. Tetrahedron 2004, 60, 6791.
9. (a) Holzer, W.; Hahn, K.; Brehmer, T.; Claramunt, R. M.; Pérez-Torralba, M. Eur.
J. Org. Chem. 2003, 7, 1209; (b) Bieringer, S.; Holzer, W. Heterocycles 2006, 68,
1825.
6. Microbiological assays
6.1. Mycobacterial strain
10. NOE interactions between benzyl protons and methyl at C3 was revealed,
whereas no NOE interactions between benzyl and p-chlorophenyl protons
were observed.
12. Manetti, F.; Corelli, F.; Biava, M.; Fioravanti, R.; Porretta, G. C.; Botta, M.
Farmaco 2000, 55, 484.
M. tuberculosis H37Rv ATCC 27294 was used in this study. It was
maintained on Löwenstein-Jensen (bioMérieux, Marcy l’Étoile,
France) agar slants until needed.
13. The weaker activity of fluoro-derivative
3 seems to be in contrast with
pharmacophoric investigations and biological data of the other N1-p-
halogen compounds. However, we hypothesised that the electron-
withdrawing fluorine atom might have an inductive effect on the
pyrazole nucleus and alteration of its electronic nature might resolve into
a loss of activity.
6.2. Antimicrobial susceptibility testing
MICs were determined by a standard twofold agar dilution meth-
od. Briefly, 1 mL of Middlebrook 7H11 agar (Becton Dickinson BBL,
Sparks, MD) supplemented with 10% oleic acid-albumin-dextrose-
catalase enrichment containing the testing compounds in 24-multi-
14. De Logu, A.; Onnis, V.; Saddi, B.; Congiu, C.; Schivo, M. L.; Cocco, M. T. J.
Antimicrob. Chemother. 2002, 49, 275.
15. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.;
Karplus, M. J. Comput. Chem. 1983, 4, 187.
16. (a) Smellie, A.; Teig, S. L.; Towbin, P. J. Comput. Chem. 1995, 16, 171; (b)
Smellie, A.; Kahn, S. D.; Teig, S. L. J. Chem. Inf. Comput. Sci. 1995, 35, 285;
(c) Smellie, A.; Kahn, S. D.; Teig, S. L. J. Chem. Inf. Comput. Sci. 1995, 35,
295.
well plates at concentrations ranging between 0.0312 and 64 lg/mL
was inoculated with 10 lL of a suspension containing M. tuberculosis
H37Rv 1.5 Â 105 cfu/mL grown on Middlebrook 7H9 broth (Difco
Laboratories, Detroit, MI) supplemented with 10% albumin-dex-