L. A. Masterson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 252–256
PBD Monomer (1a)
255
PBD Dimer (2a)
2000000
1500000
1000000
500000
1000000
750000
500000
250000
0
0
Minutes
Minutes
Figure 4. Conversion of the carbamate PBD monomer (1a) and PBD dimer (2a) prodrugs into parent PBDs (3 and 4, respectively) by CPG2 at 37 ꢁC.
Left-hand panel: h = 1a, j = 3, d = control; Right-hand panel: h = 2a, j = 4, d = control; s = mono-protected PBD dimer intermediate. Note:
control = incubation of prodrugs in the absence of CPG2; Y axis = relative areas under HPLC peaks.
gave much better cytotoxicity differentials between the
parent PBDs and prodrugs, with differentials of >7.1
and >15.4, respectively, for 1 h exposure rising to
>14.3 and >44.7 for continuous exposure. All of the
compounds examined were between 5- and 27-times
more cytotoxic on continuous exposure compared to
1 h incubation, presumably due to both increased
exposure time and hydrolysis of the prodrugs.
monomer and dimer parent PBDs upon exposure to
enzyme. As the released PBD dimer (4) and related
analogues (e.g., SJG-136, 6) are known to produce
DNA interstrand cross-links that are difficult for can-
cer cells to repair,5,6 the potential exists to develop
second-generation ADEPT prodrugs that are less
prone to the development of resistance compared to
mustard-based agents. Further work is underway to
study the behaviour of the prodrugs in human tumour
xenografts and to improve the stability of the pro-
drugs and the cytotoxicity of the released PBDs.
Finally, the carbamate monomer and dimer prodrugs
1a and 2a were incubated with 1 U of CPG2 and both
compounds were shown to be good substrates (Fig. 4).
The monomer prodrug 1a was completely converted
into the cytotoxic parent PBD 3 within 50 min with
no apparent chemical degradation observed in the as-
say buffer (100 mM Tris–HCl/260 lM ZnCl, pH 7.3)
in the absence of CPG2 over the same time period.
Similarly, the dimer prodrug 2a was completely con-
verted into the cytotoxic PBD dimer 4 in 75 min with
no chemical degradation in the absence of CPG2.
Interestingly, in the latter case, it was possible to ob-
serve the formation of an intermediate (s, Right-hand
panel, Fig. 4) thought to be the mono-protected PBD
dimer with only one glutamic acid residue cleaved. As
anticipated, this intermediate converted into the fully
de-protected PBD dimer 4 during the course of the
experiment. To confirm the potential value of these
prodrugs in ADEPT therapy, the monomer (1a) and
dimer (2a) prodrugs were incubated with CPG2 in
the presence of LS174T cells for 1 h. This reduced
the IC50 values for 1a and 2a by 7.6- and 9.0-fold,
respectively, thus confirming their transformation into
cytotoxic species.
Acknowledgments
Cancer Research UK is thanked for providing financial
support for this work (Project Grant: C180/A1066 to
DET, PWH, RHB and JAH).
References and notes
1. Thurston, D. E. The chemotherapy of cancer, 4th ed. In
Smith and WilliamsÕ Introduction to the Principles of Drug
Design & Action; Smith, H. J., Ed.; CRC Press: Boca
Raton, Florida, 2005; pp 411–522.
2. Denny, W. A. Eur. J. Med. Chem. 2001, 36, 577.
3. Niculescu-Duvaz, I.; Friedlos, F.; Niculescu-Duvaz, D.;
Davies, L.; Springer, C. J. Anti-Cancer Drug Des. 1999, 14,
517.
4. Webley, S. D.; Francis, R. J.; Pedley, R. B.; Sharma, S. K.;
Begent, R. H. J.; Hartley, J. A.; Hochhauser, D. Br. J.
Cancer 2001, 84, 1671.
5. Hartley, J. A.; Spanswick, V. J.; Brooks, N.; Clingen, P.
H.; McHugh, P. J.; Hochhauser, D.; Pedley, R. B.;
Kelland, L. R.; Alley, M. C.; Schultz, R.; Hollingshead,
M. G.; Schweikart, K. M.; Tomaszewski, J. E.; Sausville,
E. A.; Gregson, S. J.; Howard, P. W.; Thurston, D. E.
Cancer Res. 2004, 64, 6693.
6. Clingen, P. H.; De Silva, I. U.; McHugh, P. J.; Ghadessy,
F. J.; Tilby, M. J.; Thurston, D. E.; Hartley, J. A. Nucleic
Acids Res. 2005, 33, 3283.
In conclusion, these results demonstrate that it is pos-
sible to synthesize N10-protected PBD prodrugs suit-
able for CPG2-based ADEPT therapy. The prodrugs
are, in the case of the more stable carbamate deriva-
tives 1a and 2a, significantly less cytotoxic than the
parent compounds, and are good substrates for
CPG2, being rapidly converted into the cytotoxic