290
M. Silva et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 283–290
(PEG-PASP-PZA) and the carrier (PEG-PASP). The MICs of each sub-
stance for M. tuberculosis standard strains H37Ra – 25177ATCC
and H37Rv – 27294ATCC (denoted Ra, Rv) were estimated by the
microplate Alamar Blue assay, proposed by Franzblau et al. [16]:
the strains were cultured in broth containing serial dilutions of
test compounds in sealed wells in a 96-well microplate. After
5 days at 378C, a solution of Alamar Blue, a redox dye that is
reduced from blue to pink by growing M. tuberculosis cells, was
added to the wells, which were resealed and incubated for a
further 24 h. The MIC was the lowest concentration of a sub-
stance that prevented the well changing from blue to pink.
Poly(ethylene glycol)-poly(aspartic acid) block
copolymer (PEG-PASP) [7, 11, 12, 14]
PEG-PBLA (7) (4.94 g) was dissolved in 49 mL stirred chloroform
at 08C. Next, 63 mL of 0.43 mol NaOH, dissolved in a mixture of
water, 2-propanol, and methanol (1:2:2 v/v/v), was added. After
10 min reaction, the mixture was neutralized with acetic acid
and the whole poured into 65 mL of ether. The resulting precipi-
tate was filtered, redissolved in water and dialyzed against dis-
tilled water, firstly for 4 h in a dialysis membrane with exclusion
limit 1.0 kDa and then in a second, with exclusion limit 12–
14 kDa, for an equal time. The product was then lyophilized to a
white to brownish-yellow odorless semi-solid (8). Yield: 3.01 g.
Number of free carboxylic groups on the copolymer, available
for reaction with PZACH2OH, was determined as 5.8610– 4 mol
COOH per g PEG-PASP. 1H-NMR (D2O) d: 4.35 (s, CHa- & b-amide), 3.59
(m, H-2,3), 3.27 (s, H-1), 2.64 (brs, CH2 a-amide). 13C-NMR (D2O) d:
177.8 (C11), 175.6 (C8), 168.8 (C7), 70.1 (C2), 63.0 (C5), 62.0 (C1), 32.8
(C3, C6, C9), 55.2 (C5).
References
[1] World Health Organization; Global Tuberculosis Control;
WHO Report 2005; Geneva: Available in the Internet.
index.html.
Hydroxymethylpyrazinamide (PZACH2OH) [15]
Pyrazinamide (9) (6.0 g, 0.0487 mol) was suspended in 30 mL
water and 20 mL of 4 wt% aqueous K2CO3 solution added, fol-
lowed by 20 mL of 38 wt% formaldehyde (10). The mixture was
stirred for 20 h at room temperature, a further 50% of the for-
maldehyde was added and the mixture stirred for 4 h, at room
temperature and filtered. The filtrate was evaporated to dryness
at reduced pressure. The yellow solid residue was dissolved in
water-acetone (10:90 v/v), recrystallized and dried to give a
white to brownish-yellow odorless powder (11). Yield: 1.65 g
(80%); melting range 126–1288C. 1H-NMR (DMSO-d6) d: 8.94 (s, H-
2, 1H), 8.67–8.63 (d, H-4, 1H), 8.20-8.30 (d, H-3, 1H), 4.96 (s, H-6,
2H). 13C-NMR (DMSO-d6) d: 163.9 (C5), 145.6 (C4), 144.5 (C3), 144.4
(C2), 128.2 (C1), 63.4 (C6).
[2] R. Gupta, M. A. Espinal, M. C. Raviglione, Semin. Respir
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[3] D. Maher, L. Blanc, M. Raviglione, Lancet 2004, 363, 1911.
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2003, 327, 822–823.
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al., J. Control Release 2001, 74, 295–302.
Poly(ethylene glycol)-poly(pyrazinamidomethyl
[8] C. Dye, C. J. Watt, D. M. Bleed, S. M. Hosseini, M. C. Ravi-
aspartate) copolymer (PEG-PASP-PZA) [7, 11, 12, 14]
Hydroxymethylpyrazinamide (0.197 g, 0.0487 mol) was dis-
solved in 1.20 mL DMF. The condensing agent 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide hydrochloride EDC (0.82 g,
12) was added to a solution of 2.30 g PEG-PASP in 20 mL water.
The second solution was poured into the first and the ensuing
reaction was carried out at 08C for 4 h, with magnetic stirring.
Another aliquot of EDC was added and the solution stirred for
24 h, at room temperature. The solution produced was dialyzed
against acetate buffer (0.1 M, pH 4.5) for 4 h, in a membrane of
exclusion limit 1.0 kDa, and then against distilled water, for an
equal period. Finally, it was lyophilized, leaving a brownish-yel-
low odorless semi-solid (13). Yield: 1.65 g. Taking the estimated
number of free carboxylic groups in PEG-PASP to be the true
total, the percent substitution of these groups by PZACH2OH was
85.7% (4.9706610– 4 mol). 1H-NMR (D2O) d: 8.63–8.57 (l, H-14, H-
16, H-17), 5.65 (brs, H-12), 4,87 (s, CHa- & b-amide), 3.50 (m, H-2, H-3),
3.01 (s, H-1), 2.87 (brs, H-9), 2.72 (brs, H-6). 13C-NMR (D2O) d: 172.3
(C7), 170.9 (C11), 168.6 (C8), 162.7 (C4, C13), 147.6 (C17, C16), 144.8
(C15), 143.5 (C14), 69.8 (C2), 62.7 (C10), 52.2 (C1), 39.9 (C3).
glione, JAMA 2005, 293, 2767–2775.
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[15] F. P. Bonina, L. Montenegro, G. Trapani, M. Franco, G.
Liso, Int. J. Pharm. 1995, 124, 45–51.
[16] S. G. Franzblau, R. S. Witzig, J. C. Mclaughlin, P. Torres, et
al., J. Clin. Microbiol. 1998, 36, 362–366.
[17] D. Attwood, A. T. Florence, Surfactants systems: their chem-
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1983 pp. 72–117.
Biological method
Microbiological in vitro assay – This assay was performed with
the free drug, the synthesized micellar prodrug derived from it
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