J. F. Marquez Ruiz et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1693–1698
1697
7.0×10-7
2.0×10-5
1.5×10-5
1.0×10-5
5.0×10-6
0
A
B
6.0×10-7
5.0×10-7
4.0×10-7
3.0×10-7
P
P
2.0×10-7
1.0×10-7
0
Budesonide
3
Celecoxib
5
6
Figure 4. (A) Apparent permeability coefficient of 5, 6 and celecoxib; (B) Apparent permeability coefficient transport of 3 and budesonide. There was no evidence of transport
of 4 under similar conditions.
50
40
30
20
10
0
50
40
30
20
10
0
A
B
0
250
500
750 1000 1250 1500
0
250
500
750 1000 1250 1500
Time [min]
Time [min]
Figure 5. (A) Stability of 1 (j), 2 (D D), 3 (ꢀ), 4 (s), 5 (h) and 6
), 3 (ꢀ), 4 (s), 5 (h) and 6 (N) in simulated human gastric juices at 37 °C.22 (B) Stability of compounds 1 (j), 2 (
(N) in simulated human duodenal fluid at 37 °C.22
development rivals in part because of their permeability character-
istics. Therefore we were confident that the relatively small struc-
tural changes involved in the prodrug design would be sufficient to
significantly attenuate passive diffusion potential. Absorptive
transport from apical to basolateral side (AP > BL) was evaluated
for appearance of the test compound and in the case of the pro-
drugs, the potential metabolic byproducts. Aliquots were collected
after 0, 30, 60, and 120 min from the acceptor reservoir and fresh
buffer was added to maintain the initial volume. The donor solu-
tion was also collected after 120 min. Caffeine was tested in two
wells in the AP > BL direction as a routine control. Trans-epithelial
byproducts of the reduction/cyclization process under simulated
colonic conditions; (ii) the potential for elaboration of the linker
unit in order to influence (i) as well as pharmaceutical properties
such as stability and permeability; the rates of reduction of the ni-
tro group and its electronic and steric control.24
References and notes
1. Krishnaiah, Y. S.; Khan, M. A. Pharm. Dev. Technol. 2012, 17, 521.
2. Xiao, B.; Merlin, D. Expert Opin. Drug Deliv. 2012, 9, 1393.
3. Patel, M.; Amin, A. Crit. Rev. Ther. Drug Carrier Syst. 2011, 28, 489.
4. Vaidya, A.; Agarwal, A.; Jain, A.; Agrawal, R. K.; Jain, S. K. Curr. Pharm. Des. 2011,
17, 1108.
electric resistance (TEER) was over 350
X
cm2 in all wells used in
the experiments prior to the addition of the test compounds.21
Compounds 3, 5, 6 possessed significantly lower permeability
than the parents celecoxib and budesonide (Fig. 4). No transport
of budesonide prodrug 4 could be detected. This data indicates that
the compounds would be able to resist absorption and transit the
intestinal tract if delivered orally. Nitroreductase-triggered cycliza-
tion and drug release may therefore be a generally effective ap-
proach to achieving colorectal drug targeting. The prodrugs were
processed in the presence of C. perfingens present in the human co-
lon, releasing celecoxib, prednisolone or budesonide. The stability
of the candidate prodrugs in this study was tested using the USP
gastric and intestinal fluid model, which mimics the enzymatic
and chemical conditions in the stomach and small intestine.22
Amounts of remaining compound was assessed using HPLC. The
compounds were found to be stable over the expected orocaecal
transit time (Fig. 5).23 Finally, they are predicted by the standard
Caco-2 permeability assay to be poorly absorbed relative to their
parents suggesting that they might arrive as intact substrates for
reductase activity associated with the microflora. Several aspects
deserve further study: (i) the identity and biological profile of
5. Dhaneshwar, S. S.; Vadnerkar, G. Curr. Top. Med. Chem. 2011, 11, 2318.
6. Varshosaz, J.; Emami, J.; Fassihi, A.; Tavakoli, N.; Minaiyan, M.; Ahmadi, F.;
Mahzouni, P.; Dorkoosh, F. Int. J. Colorectal Dis. 2010, 25, 1159.
7. Varshosaz, J. Expert Opin. Drug Deliv. 2010, 9, 509.
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Gentili, G.; Setchell, K. D.; O’Connell, N.; Pellicciari, R.; Clerici, C. Anticancer Res.
2009, 29, 4971.
9. Mishra, A.; Agrawal, S.; Pathak, K. Drug Deliv. 2012, 19, 102.
10. Rodrigues, C. M.; Kren, B. T.; Steer, C. J.; Setchell, K. D. Gastroenterology 1995,
109, 1835.
11. 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.
12. Ruiz, J. F.; Kedziora, K.; Keogh, B.; Maguire, J.; Reilly, M.; Windle, H.; Kelleher, D.
P.; Gilmer, J. F. Bioorg. Med. Chem. Lett. 2011, 21, 6636.
13. Hao, J.; Ge, F.; Liu, Y.; Qian, Y. Peop. Rep. China Patent CN 1760173, 2006.
14. Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394.
15. Spectroscopic data for 5. 1H NMR (CDCl3) d: 8.70 (1H, s., 8.10 (1H, d, J 8.52 Hz),
8.05 (2H, d, J 8.56 Hz), 7.61 (1H, t, J 7.52 Hz), 7.51 (3H, m), 7.38 (2H, d, J
7.52 Hz), 7.27 (1H, s), 7.16 (2H, d, J 7.52 Hz), 7.12 (2H, d, J 8.04 Hz), 6.74 (1H, s),
3.89 (2H, s), 2.38 (3H, s). 13C NMR (CDCl3) d: 166.37, 147.89, 144.93, 143.14,
139.38, 136.94, 133.72, 133.28, 129.35, 129.09, 128.93, 128.28, 127,55, 125.15,
125.05, 124.71, 105.98, 41.14, 29.24, 20.87. Expected (MꢀH+) = 545.1151,
Found (MꢀH+) = 545.1113.
16. Ruiz, J. F.; Kedziora, K.; Windle, H.; Kelleher, D. P.; Gilmer, J. F. J. Pharm.
Pharmacol. 2011, 63, 806.