W. Kemnitzer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6259–6264
6263
erocycle is preferred in the 4-position. In comparison, the piperi-
dine analog 2j was found to be inactive up to 10 lM for the three
2-(2-pyridyl)pyrimidines with different substituents in the 4-anili-
no group.27
cell lines tested.
In conclusion, we have discovered and explored the SAR of the
apoptosis inducing 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines by
modifying the 1 and 3-positions. It was found that small electron
withdrawing groups at the 4-position of the 1-benzoyl group like
F and Cl are preferred to maintain potency and selectivity in the
caspase activation assay. Substitution at the 2- or 3-positions of
the benzoyl group also resulted in active compounds. In addition,
the phenyl ring could be replaced by a 2-pyridyl group. The 3-cya-
no group was found to be important for activity. Interestingly, sub-
stitution at the 4-position of the 1-benzoyl group with an
imidazole (2k) or pyrazole (2l) group resulted in highly active com-
pounds with a different caspase activation profile. These com-
pounds were broadly active and induced apoptosis by inhibiting
tubulin polymerization. In addition, hydroxyl analogs were also ac-
tive offering the potential for improving aqueous solubility.
Through SAR studies of the 1-benzoyl-3-cyanopyrrolo[1,2-a]quin-
olines, compound 2k was identified, which has low nanomolar po-
tency in both the caspase activation assay and growth inhibition
assay and is significantly more potent than the initial hit 2a. Addi-
tional SAR studies of the 4-, 5-, 6-, 7-, and 8-positions of 1-benzoyl-
3-cyanopyrrolo[1,2-a]quinolines will be reported in future
publications.
In an effort to improve the solubility profile, the more rigid car-
bonyl linker of the benzoyl group was reduced to a more flexible
hydroxyl group. Analog 3a was similar in potency to 2b, indicating
that changing the carbonyl to a hydroxyl group is tolerated. Simi-
larly, the hydroxyl analog 3b was about as active as the carbonyl
analog 2i, while analog 3c was 4-fold less active than 2l.
The SAR at the 3-position of the pyrrolo[1,2-a]quinoline struc-
ture was also investigated. Replacing the 3-cyano group of 2c with
an acetyl group gave the analog 4, which was found to be inactive
in the caspase activation assay up to 10 lM. Additionally, analog 5,
with the 3-cyano group of 2n replaced with an ethyl ester group,
was found to be inactive, indicating that the cyano group at the
3-position might be essential for activity.
Selected compounds were also tested by the traditional cell
growth inhibition assay (GI50) to confirm that the active com-
pounds can inhibit tumor cell growth. The growth inhibition as-
says in T47D, HCT116, and SNU398 cells were run in a 96-well
microtiter plate as described previously.27 The GI50 values are
summarized in Table 2. Compound 2a was found to have selec-
tivity similar to the EC50 caspase assay with GI50 values of 0.070,
5.8, and 2.0 lM in T47D, HCT116, and SNU398 cells, respectively.
In general, compounds2b, 2g, 2i, 2l, and 2m were more sensitive
to T47D and SNU398 cells, than to HCT116 cells. Compounds 2b
and 2i both had GI50 values <100 nM in the T47D cells. Com-
pound 2k, which was the most active compound in the caspase
assay, was also the most active in the GI50 assay with values of
References and notes
1. Varmus, H. Science 2006, 312, 1162.
2. Haskell, C. M. In Cancer Treatment, 5th ed.; W. B. Saunders Company:
Philadelphia, PA, 2001; Chapter 1.
3. Eckhardt, S. Curr. Med. Chem. Anti-Cancer Agents 2002, 2, 419.
4. Lu, M. C. In Cancer Chemotherapeutic Agents; Foye, W. O., Ed.; American
Chemical Society: Washington, DC, 1995, p 345.
0.041, 0.065, and 0.059
respectively.
lM for T47D, HCT116, and SNU398 cells,
Compounds 2a and 2k were characterized by cell cycle analy-
sis20 in HCT116 cells and found to arrest cell in G2/M and induce
apoptosis. Since tubulin inhibitors are known to arrest cells in
G2/M, we tested several compounds in a tubulin polymerization
assay31 in an attempt to differentiate the mechanism of action of
the broad active vs. the selective compounds. Compound 2a, which
was active against breast cancer cells but much less active against
several other cancer cell lines, was found to be inactive in the tubu-
5. Altmann, K. H. Curr. Opin. Chem. Biol. 2001, 5, 424.
6. Wartmann, M.; Altmann, K. H. Curr. Med. Chem. Anti-Cancer Agents 2002, 2, 123.
7. O’Dwyer, M. E.; Druker, B. J. Curr. Cancer Drug Targets 2001, 1, 49.
8. Nutt, J. E.; Lazarowicz, H. P.; Mellon, J. K.; Lunec, J. Br. J. Cancer 2004, 8, 1679.
9. Dowell, J.; Minna, J. D. Nat. Rev. Drug Disc. 2005, 5, s14.
10. Moy, B.; Goss, P. E. Oncologist 2006, 10, 1047.
11. Faivre, S.; Demitri, G.; Sargent, W.; Raymond, E. Nat. Rev. Drug Disc. 2007, 6,
734.
12. Reed, J. C. J. Clin. Oncol. 1999, 17, 2941.
13. Mehlen, P.; Puisieux, A. Nat. Rev. 2006, 6, 449.
14. Vial, J. P.; Belloc, F.; Dumain, P.; Besnard, S.; Lacombe, F.; Boisseau, M. R.;
Reiffers, J.; Bernard, P. Leuk. Res. 1997, 21, 163.
15. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43, 305.
16. Thornberry, N. A. Chem. Biol. 1998, 5, 97.
17. Salvesen, G. S.; Dixit, V. M. Cell 1997, 91, 443.
18. Cai, S. X.; Zhang, H. Z.; Guastella, J.; Drewe, J.; Yang, W.; Weber, E. Bioorg. Med.
Chem. Lett. 2001, 11, 39.
19. Zhang, H. Z.; Kasibhatla, S.; Guastella, J.; Drewe, J.; Tseng, B.; Cai, S. X.
Bioconjugate Chem. 2003, 14, 458.
20. Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang, H.; Wang, Y.; Zhao, J.; Jia, S.; Herich,
J.; Labrecque, D.; Storer, R.; Meerovitch, K.; Bouffard, D.; Rej, R.; Denis, R.; Blais,
C.; Lamothe, S.; Attardo, G.; Gourdeau, H.; Tseng, B.; Kasibhatla, S.; Cai, S. X. J.
Med. Chem. 2004, 47, 6299.
21. Kasibhatla, S.; Gourdeau, H.; Meerovitch, K.; Drewe, J.; Reddy, S.; Qiu, L.; Zhang,
H.; Bergeron, F.; Bouffard, D.; Yang, Q.; Herich, J.; Lamothe, S.; Cai, S. X.; Tseng,
B. Mol. Cancer Ther. 2004, 3, 1365.
lin polymerization assay at up to 50 lM. Compounds 2l and 2k,
both of which were broadly active against the three cancer cell
lines tested, were found to be active in the tubulin polymerization
assay, with IC50 values of 1 and 5 lM, respectively, indicating that
compounds 2l and 2k and other broadly active compounds in this
series most probably induce apoptosis through inhibition of tubu-
lin polymerization. These data confirmed that with different sub-
stituents in the 4-position of the 1-benzoyl group, the selective
compounds and non-selective compounds have different mecha-
nism of actions. We have observed previously a similar change
from selective compounds to non-selective compounds and change
of mechanism of action for a series of apoptosis inducing 4-anilino-
22. Gourdeau, H.; Leblond, L.; Hamelin, B.; Desputeau, C.; Dong, K.; Kianicka, I.;
Custeau, D.; Bourdeau, C.; Geerts, L.; Cai, S. X.; Drewe, J.; Labrecque, D.;
Kasibhatla, S.; Tseng, B. Mol. Cancer Ther. 2004, 3, 1375.
23. Zhang, H. Z.; Kasibhatla, S.; Wang, Y.; Herich, J.; Guastella, J.; Tseng, B.; Drewe,
J.; Cai, S. X. Bioorg. Med. Chem. 2004, 12, 309.
24. Kasibhatla, S.; Jessen, K. A.; Maliartchouk, S.; Wang, J. Y.; English, N. M.; Drewe,
J.; Qiu, L.; Archer, S. P.; Ponce, A. E.; Sirisoma, N.; Jiang, S.; Zhang, H. Z.; Gehlsen,
K. R.; Cai, S. X.; Green, D. R.; Tseng, B. Proc. Natl. Acad. Sci. U.S.A. 2005, 34, 12095.
25. Zhang, H. Z.; Kasibhatla, S.; Kuemmerle, J.; Kemnitzer, W.; Ollis-Mason, K.; Qiu,
L.; Crogan-Grundy, C.; Tseng, B.; Drewe, J.; Cai, S. X. J. Med. Chem. 2005, 48,
5215.
26. Jessen, K.; English, N.; Wang, J.; Qui, L.; Brand, R.; Maliartchouk, S.; Drewe, J.;
Kuemmerle, J.; Zhang, H. Z.; Gehlsen, K.; Tseng, B.; Cai, S. X.; Kasibhatla, S. Mol.
Cancer Ther. 2005, 4, 761.
Table 2
Inhibition of cell growth of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines.
Compound
GI50
HCT116
(l
M)a
T47D
SNU398
2.0 0.50
0.12 0.004
0.42 0.086
0.14 0.013
0.059 0.009
0.036 0.007
0.73 0.13
2a
2b
2g
2i
2k
2l
0.070 0.015
0.071 0.008
0.47 0.056
0.075 0.014
0.041 0.008
0.072 0.026
0.087 0.013
5.8 0.13
0.49 0.008
1.2 0.11
0.25 0.038
0.065 0.015
0.24 0.10
1.6 0.18
2m
27. Sirisoma, N.; Kasibhatla, S.; Nguyen, B.; Pervin, A.; Wang, Y.; Claassen, G.;
Tseng, B.; Drewe, J.; Cai, S. X. Bioorg. Med. Chem. 2006, 14, 7761.
28. Wei, X.; Hu, Y.; Li, T.; Hu, H. J. Chem. Soc., Perkin Trans. 1 1993, 2487.
29. Zhang, X.; Cao, W.; Wei, X.; Hu, H. Synth. Commun. 1997, 8, 1395.
a
Data are the mean of three or more experiments and are reported as
mean standard error of the mean (SEM).