7652
J. Gavin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7647–7652
electron donating effect on the benzene ring.33 This substituent ef-
fect appears to promote disproportionation of the hydrazine inter-
mediate to the parent amine compounds akin to that of Group A
compounds, while still imparting enough stability to permit its
detection via HPLC.
stabilizing their hydrazine intermediates might be a useful strat-
egy in reducing the toxic liability related to their amino reduction
products.
References and notes
Several other studies have shown insight into the mechanistic
features of azoreductases of species which differ from that of the
colonic anaerobe C. perfringens. Studies on mixed aerobic/anaero-
bic rabbit liver microsomal P450 azoreductase have reported that
polar electron-donating substituents are obligatory for the reduc-
tion of azo dyes,34 and that compounds without such constituents
will not bind to cytochrome P450 or undergo any reduction. Fer-
rous cytochrome P450 enzymes have shown a one-electron reduc-
tion mechanism involving a free-radical intermediate.35 Flavin-
dependent enzymes such as C. perfringens have shown a two-elec-
tron mechanism forming no free-radical intermediates, and have
shown different substrate specificity to flavin-independent
enzymes.32
A separate study on the mechanism of azoreduction of balsalaz-
ide by aerobic Pseudomonas aeruginosa proposed that tautomeriza-
tion of azo substrates to the hydrazone form is necessary for
azoreduction.14 This phenomenon has been demonstrated in azo
compounds with an electron donor group36 such as the Group A
compounds in this Letter. Group B compounds however do not
contain a protic donor group on either phenyl ring and therefore
are unable to tautomerize. It can therefore be assumed that the
hydrazine is generated by direct transfer of hydride to the azo
group in the first step, and this stable intermediate is not subject
to further enzymatic electron transfer.
1. Chung, K. T.; Stevens, S. E., Jr.; Cerniglia, C. E. Crit. Rev. Microbiol. 1992, 18, 175.
2. Stolz, A. Appl. Microbiol. Biotechnol. 2001, 56, 69.
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2000, 66, 187.
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FEMS Microbiol. Lett. 2004, 236, 129.
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Biotechnol. 2007, 76, 1271.
13. Wang, C. J.; Laurieri, N.; Abuhammad, A.; Lowe, E.; Westwood, I.; Ryan, A.; Sim,
E. Acta Cryst. 2010, F66, 2.
14. Ryan, A.; Laurieri, N.; Westwood, I.; Wang, C. J.; Lowe, E.; Sim, E. J. Mol. Biol.
2010, 400, 24.
15. Ruiz, J. F.; Radics, G.; Windle, H.; Serra, H. O.; Simplicio, A. L.; Kedziora, K.;
Fallon, P. G.; Kelleher, D. P.; Gilmer, J. F. J. Med. Chem. 2009, 52, 3205.
16. Compound 1c. Mp: 184–186 °C. IRvmax (KBr): 3468.24, 1638.01, 1618.44 and
1384.43 cmꢁ1 1H NMR d (CD3OD): 8.21 (2H, d, J 8.28 Hz), 7.99 (2H, d, J
.
8.28 Hz), 7.74 (1H, d, J 8.04 Hz), 7.49 (3H, m), 7.36 (1H, m), 6.29 (1H, dd, J 10.04
and 1.76 Hz), 6.02 (1H, s) 4.99 (1H, d, J 17.56 Hz, one hydrogen is masked by
the water peak), 4.42 (1H, s), 3.53 (2H, t, J 7.76 Hz), 2.85 (2H, t, J 8.52 Hz) 2.69–
0.94 (19H, prednisolone envelope) 1.71 (9H, s). 13C NMR d (MeOD): 205.73
(C@O, C-20), 187.61 (C@O, C-3), 173.43 (C@O, C-22), 172.71 (CH, C-1), 171.45
(C, C-5), 165.79 (C, C-100), 158.83 (C@O, C-130), 154.48 (C, C-20), 149.95 (C, C-
70), 145.41 (C, C-10), 140.81 (CH, C-50), 131.21 (CH, C-60), 130.46 (CH, C-90),
130.21 (CH, C-110 and C-30), 127.06 (CH, C-2), 126.41 (CH, C-4), 122.18 (CH, C-
80), 121.08 (CH, C-120), 114.93 (CH, C-40), 89.08 (C, C-17), 69.37 (CH, C-11),
67.94 (CH2, C-21), 55.94 (CH, C-9), 51.44 (CH, C-14), CD3OD residual peak is
masking the peak of C, C-13, 44.68 (C, C-10), 38.85 (CH2, C-12), 35.76 (CH2, C-
23), 34.21 (CH2, C-6), 33.25 (CH2, C-7), 31.81 (CH2, C-16), 31.26 (CH, C-8), 26.79
(CH2, C-24), 23.45 (CH2, C-15), 20.22 (CH3, C-19), 15.91(CH3, C-18). HRMS:
Found: (MꢁH)+ = 641.2863, Required: (MꢁH)+ = 641.2850.
The presence and prevalence of the anaerobic C. perfringens in
the human colon makes it an important organism as an in vitro
model of azo prodrug activation. Despite their widespread applica-
tion, there is little published literature on the SAR of azoreduction
by colonic microflora. The observations in this Letter were made
possible because of two differences between our approach and that
generally adopted in the literature:
17. Ruiz, J. F. M.; Kedziora, K.; Windle, H.; Kelleher, D. P.; Gilmer, J. F. J. Pharm.
Pharmacol. 2011, 63, 806.
The use of HPLC to separate the intermediate from the reaction
product. Colorimetric methods have been reported for monitoring
substrate consumption by C. perfringens.37 However this method of
monitoring substrate consumption is not ideal to distinguish cases
that stop at the hydrazo stage from those that proceed to aniline
formation.10,29
18. Khan, A. A.; Nawaz, M. S.; Robertson, L.; Khan, S. A.; Cerniglia, C. E. Mol. Cell
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19. Rafii, F.; Franklin, W.; Cerniglia, C. E. Appl. Environ. Microbiol. 1990, 56, 2146.
20. Rafii, F.; Coleman, T. J. Basic Microbiol. 1999, 39, 29.
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22. Rafii, F.; Cerniglia, C. E. Environ. Health Perspect. 1995, 103, 17.
23. Patai, S. The Chemistry of the Hydrazo, Azo and Azoxy Groups; Wiley, 1997.
24. Campbell, N.; Henderson, A. W.; Taylor, D. J. Chem. Soc. 1953, 1281.
25. Leriche, G.; Budin, G.; Brino, L.; Wagner, A. Eur. J. Org. Chem. 2010, 4360.
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28. Nam, S.; Renganathan, V. Chemosphere 2000, 40, 351.
1. The use of substrates lacking an electron-donating group. Azo
compounds usually present an electron-donating substituent,
as this is required for diazonium coupling of the precursors.
The nitroso-based chemistry permitted the synthesis of substi-
tuted azobenzenes without mesomeric donors.
29. Saphier, S.; Karton, Y. J. Pharm. Sci. 2010, 99, 804.
30. Schellen, M.; Steinmet, R. Helv. Chim. Acta 1969, 52, 431.
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The inability of C. perfringens azoreductase activity to com-
pletely reduce azo compounds lacking an electron donating group
on the phenyl ring has some implications for the design of azo-
based prodrugs. It would be interesting to know if these limitations
are shared by other bacteria in the colonic microflora. There are
additional consequences regarding the toxicology of azo dyes as
37. Semdé, R.; Pierre, D.; Geuskens, G.; Devleeschouwer, M.; Moës, A. J. Int. J.
Pharm. 1998, 161, 45.