M. Biava et al. / European Journal of Medicinal Chemistry 44 (2009) 4734–4738
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silica gel and petroleum ether/ethyl acetate (3:1 v/v). The eluates
were combined after TLC control and the solvent was removed to
give 2a–h as solids in satisfactory yield. Re-crystallization from
diethyl ether gave the required products. Structure of the final
compounds was confirmed by observing the disappearance of the
singlet of the proton at the position 3 of the pyrrole (6.09 ppm) and
the appearance of the singlet corresponding to the methylene
group at 3.50 ppm [8a]. The elemental analyses for compounds 2a–
h are reported in Table 2 (Supporting Information).
5.3. 2-Ethyl-1-(4-methylphenyl)–3-(thiomorpholin-4-yl)-methyl-
5-(4-fluorophenyl)-1H-pyrrole (2b)
Mp 165 ꢀC (yield 45%); 1H NMR (CDCl3) 7.26 (m, 4H), 7.16 (m,
2H), 7.00 (m, 2H), 6.27 (s, 1H), 3.46 (s, 2H), 2.78 (s broad, 4H), 2.71 (s
broad, 4H), 2.25 (q, 2H), 2.18 (m, 3H), 0.85 (t, 3H). 13C NMR (CDCl3)
160.18 (d, 1C), 139.25 (s, 1C), 135.90 (s, 1C), 132.17 (s, 1C), 129.53 (s,
1C), 129.49 (s, 1C), 128.99 (s, 2C), 128.73 (s, 2C), 127.64 (s, 1C), 114.91
(s, 2C), 114.69 (s, 2C), 110.58 (s, 1C), 55.44 (s, 2C), 54.89 (s, 1C), 28.17
(s, 2C), 18.02 (s, 2C), 14.87 (s, 1C). Anal. (C24H27FN2S) C, H, N, S, F.
5.4. Microbiology
5.4.1. Compounds
Compounds 2b–h and reference drugs were dissolved in DMSO
at
a concentration of 5 mg/mL and stored cold until used.
Compound 2a was dissolved in ethanol at 6 mg/mL.
5.4.2. Antimycobacterial activity
Compounds were preliminarily assayed according to protocol
already described [8b], following a standard twofold agar dilution
method in Middlebrook 7H11 agar medium (Difco) containing 10%
of OADC (oleic acid, albumin and dextrose complex) [12].
Compounds with better preliminary activity were tested toward
M. tuberculosis CIP 103471, M. tuberculosis H37Rv ATCC 27294,
rifampicin-resistant M. tuberculosis ATCC 35838, and atypical
mycobacteria, such as M. avium CIP 103317. In all cases, MIC value for
each compound was determined for each mycobacterial strain.
Further details are in Supporting Information.
Fig. 1. Graphical representation of the superposition mode of 2a (green) and 1a (red)
into the pharmacophoric model. Side chains at positions 2 and 3 of 2a are oriented in
opposite directions with respect to the pyrrole plane and are only able to partially
satisfy the hydrogen bond acceptor group (green spheres) and the directionality
constraint imposed by one of the aromatic ring features (orange spheres, bottom right
corner). The hydrophobic region (cyan sphere) is filled by the p-Cl group of both
compounds, as well as the other aromatic ring feature (orange spheres, middle left).
5.4.3. Cytotoxic activity assays
further efforts are planned for a structural optimization of this class
of compounds.
Cytotoxic activity assays were performed in Vero cells to
determine the maximum non-toxic dose defined as the highest
drug concentration showing no morphological changes as deter-
mined microscopically by the observation at 72 h of incubation.
5. Experimental section
5.1. Chemistry
5.5. Computational details
The general procedure for the preparation of hexane-1,4-diones
5a–e and 1,5-diarylpyrroles 6a–h is described in Supporting
Information.
The QSARþ module of Cerius2 [13] was used to calculate
Alog P98 values of the studied compounds. Such a descriptor is an
implementation of the atom type-based Alog P method using the
latest published set of parameters [14].
5.2. General procedure for the preparation of compounds 2a–h
Acknowledgements
Following the Mannich reaction, to a stirred solution of an
appropriate pyrrole 6 (5.6 mmol) in acetonitrile (20 mL), a mixture
of thiomorpholine (0.57 g, 5.6 mmol) or N-methylpiperazine
(0.56 g, 5.6 mmol), formaldehyde (0.18 g, 5.6 mmol) (40% in water)
and 5 mL of acetic acid, was added dropwise. After the addition was
complete, the mixture was stirred at room temperature for 1 h. The
mixture was then treated with a solution of sodium hydroxide
(20%, w/v) and extracted with ethyl acetate. The organic extracts
were combined, washed with water and dried. After removal of
solvent, the residue was purified by column chromatography, using
`
Financial support from the Italian Ministero dell’Universita e
della Ricerca (PRIN 2005037820) and CARIPLO (Rif. 2006.0880/
10.8485 Bando 2006) is gratefully acknowledged.
Appendix A. Supplementary information
Supplementary data associated with this article can be found, in