Synthesis and biological evaluation of 10,11-methylenedioxy-14- azacamptothecin

Article abstract of DOI:10.1021/ol0611604

10,11-Methylenedioxy-14-azacamptothecin, a potent analogue of the antitumor agent camptothecin (CPT), has been prepared via a key condensation between AB and DE ring precursors. The biological testing of this compound validated a strategy for modulation of the off-rate of camptothecin analogues from the topoisomerase-DNA-CPT ternary complex via structural modification.

Full text of DOI:10.1021/ol0611604

ORGANIC
LETTERS
2006
Vol. 8, No. 16
3513-3516
Synthesis and Biological Evaluation of
10,11-Methylenedioxy-14-azacamptothecin
Mark A. Elban, Wenyue Sun, Brian M. Eisenhauer, Rong Gao, and
Sidney M. Hecht*
Departments of Chemistry and Biology, UniVersity of Virginia,
CharlottesVille, Virginia 22904
sidhecht@Virginia.edu
Received May 11, 2006
ABSTRACT
10,11-Methylenedioxy-14-azacamptothecin, a potent analogue of the antitumor agent camptothecin (CPT), has been prepared via a key
condensation between AB and DE ring precursors. The biological testing of this compound validated a strategy for modulation of the off-rate
of camptothecin analogues from the topoisomerase−DNA−CPT ternary complex via structural modification.
Camptothecin (CPT, 1) (Figure 1) is an alkaloid first isolated
from extracts of Camptotheca acuminata;1 the compound
exhibits potent antitumor activity but has a complicated
history of clinical development due to its poor aqueous
solubility.2 Mechanistically, CPT has been shown to function
by stabilizing the covalent binary complex between type I
DNA topoisomerase and its DNA substrate.3 These initial
findings led to a flurry of activity which has resulted in a
number of interesting CPT analogues, two of which are
marketed as anticancer agents and several of which are in
clinical trials.4
I poison.6 Accordingly, it was surprising to find that luotonin
A (2) (Figure 1), which contains nitrogen at position 14 of
the D ring and lacks the E ring lactone altogether, also acted
as a topoisomerase I poison.7 Accordingly, our laboratory
prepared and tested luotonin A, CPT, and rosettacin (struc-
tural homologue of luotinin A with a CH at position 14, 3).
Although we were able to confirm the previously reported
weak ability of rosettacin to act as a topoisomerase I poison,8
it was found that the cytotoxicity of rosettacin was not
topoisomerase I dependent.9 Thus, the presence of a CH
group rather than a nitrogen at position 14 in luotonin A
Biochemical studies of CPT revealed that the E ring was
essential for stabilization of the topoisomerase I-DNA
covalent binary complex.5 Moreover, removal of the C20-
OH group or inversion of the stereochemistry at that center
proved detrimental to the activity of CPT as a topoisomerase
(1) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A.
T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888.
(2) (a) Moertel, C. G.; Schutt, A. J.; Reitemeier, R. J.; Han, R. G. Cancer
Chemother. Rep. 1972, 56, 95. (b) Gottlieb, J. A.; Luce, J. K. Cancer
Chemother. Rep. 1972, 56, 103.
(3) (a) Hsiang, Y.-H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol. Chem.
1985, 260, 14873. (b) Hsiang, Y.-H.; Liu, L. F.; Wall, M. E.; Wani, M. C.;
Nicholas, A. W.; Manikumar, G.; Kirschenbaum, S.; Silber, R.; Potmesil,
M. Cancer Res. 1989, 49, 4385. (c) Covey, J. M.; Jaxel, C.; Kohn, K. W.;
Pommier, Y. Cancer Res. 1989, 49, 5016.
(4) (a) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Bioorg. Med. Chem.
2004, 12, 1585. (b) Rahier, N. J.; Thomas, C. J.; Hecht, S. M. In Antitumor
Agents from Natural Sources; Cragg, G. M., Kingston, D. G. I., Newman,
D. J., Eds.; CRC Press: Boca Raton, 2005; pp 357-381.
Figure 1. Structures of CPT and related analogues.
10.1021/ol0611604 CCC: $33.50
© 2006 American Chemical Society
Published on Web 07/12/2006

actually eliminated its activity as a topoisomerase I-dependent
cytotoxin. A potential rationalization for these results is
possible based on a crystal structure of a topoisomerase
I-DNA complex containing a CPT analogue.10 In this
structure, a hydrogen bond network exists between residues
Asp533 and Arg364 of topoisomerase I and the C20-OH
group of CPT, thus creating a possible steric clash between
the clustered C14-H and C20-OH/ethyl groups.
To test this hypothesis, the synthesis and biochemical
evaluation of the water-soluble 14-azaCPT (4) was carried
out.9,11 It was found that 4 stabilized the topoisomerase
I-DNA complex at the same sites as CPT and was cytotoxic
with a similar but somewhat greater IC50 value. Further, it
was also shown that 14-azaCPT mediated inhibition of DNA
relaxation more effectively than CPT. Critically, 4 possessed
a faster off-rate from the ternary complex than CPT. It
appears that replacing the C14-H group with N does
improve the ability to form the ternary complex while
concomitantly reducing the lifetime of the formed complex,
thus reducing the cytotoxic effects of the resulting analogue.
discrepancy is due to differences in the off-rates of individual
CPTs from the formed ternary complexes; those CPT
analogues having slower off-rates tend to be more cytotoxic.
Given these interesting biological results, we hypothesized
that altering structural features in different regions of the
molecule could provide a way to modulate the off-rate.
Previously, it had been shown that the 10,11-methylenedioxy
(MDO) group increases the stability of the topoisomerase
I-DNA complex, thus diminishing the off-rate.14a By
incorporating this structural feature into the 14-aza core, it
seemed that it might be possible to maintain the stabilization
and inhibitory properties of CPT while decreasing the off-
rate, thus making a more effective topoisomerase I poison
and anticancer therapeutic candidate. The synthesis and initial
biological testing of 10,11-MDO-14-azaCPT (5) are pre-
sented herein.
The synthesis of 10,11-MDO-14-azaCPT is outlined in
Schemes 1 and 2. The required dibromide 9 was prepared
Previously, it was found that the key event leading to cell
death was the persistence of the ternary complex, which can
be converted to a double-strand break in DNA as a
consequence of interaction with a moving replication fork.12
Numerous studies now support the thesis that failure to
resolve the formed protein-DNA complex, i.e., persistence
of the complex, is correlated to the development of a
cytotoxic response.13 Although the concentration of topoi-
somerase I-DNA-CPT ternary complexes formed at equi-
librium logically constitutes the source of the cytotoxic
response resulting from treatment with CPT (analogues), it
has been shown convincingly that CPT analogues that
promote ternary complex formation to comparable extents
can nonetheless differ in the cytotoxic response that they
promote.14 The available data argue that much of the apparent
Scheme 1. Synthesis of Dibromide 9
in four steps from commercially available 3,4-(methylene-
dioxy)aniline which was initially acetylated to afford 6 in
(5) (a) Hutchinson, C. R. Tetrahedron 1981, 37, 1047. (b) Hertzberg, R.
P.; Caranfa, M. J.; Holden, K. G.; Jakas, D. R.; Gallagher, G.; Mattern, M.
R.; Mong, S.-M.; Bartus, J. O.; Johnson, R. K.; Kingsbury, W. D. J. Med.
Chem. 1989, 32, 715.
Scheme 2. Completion of the Synthesis of
10,11-MDO-14-azaCPT (5)
(6) (a) Jaxel, C.; Kohn, K. W.; Wani, M. C.; Wall, M. E.; Pommier, Y.
Cancer Res. 1989, 49, 1465. (b) Hertzberg, R. P.; Caranfa, M. J.; Hecht, S.
M. Biochemistry 1989, 28, 4629. (c) Wang, X.; Zhou, X.; Hecht, S. M.
Biochemistry 1999, 38, 4374.
(7) Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B. M.; Hecht, S. M. J.
Am. Chem. Soc. 2003, 125, 13628.
(8) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Staker, B. L.; Stewart, L.; Cushman, M. J. Med. Chem. 2003, 46, 3275.
(9) Cheng, K.; Rahier, N. J.; Eisenhauer, B. M.; Thomas, S. J.; Gao, R.;
Hecht, S. M. J. Am. Chem. Soc. 2005, 127, 838.
(10) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin,
A. B., Jr.; Stewart, L. Proc. Natl. Acad. U.S.A. 2002, 99, 15387.
(11) Rahier, N. J.; Cheng, K.; Gao, R.; Eisenhauer, B. M.; Hecht, S. M.
Org. Lett. 2005, 7, 835.
(12) (a) Hsiang, Y.-H.; Lihou, M. G.; Liu, L. F. Cancer Res. 1989, 49,
5077. (b) Zhang, H.; D’Arpa, P.; Liu, F. F. Cancer Cells 1990, 2, 23.
(13) (a) Wu, J.; Yin, M.-B.; Hapke, G.; Toth, K.; Rustum, Y. M. Mol.
Pharmacol. 2002, 61, 742. (b) Desai, S. D.; Zhang, H.; Rodriguez-Bauman,
A.; Yang, J.-M.; Wu, X.; Gounder, M. K.; Rubin, E. H.; Liu, L. F. Mol.
Cell. Biol. 2003, 23, 2341. (c) Zhang, H.-F.; Tomida, A.; Koshimizu, R.;
Ogiso, Y.; Lei, S.; Tsuruo, T. Cancer Res. 2004, 64, 1114. (d) Adachi, N.;
So, S.; Koyama, H. J. Biol. Chem. 2004, 279, 37343. (e) van Waardenburg,
R. C. A. M.; deJong, L. A.; Eijndhoven, M. A. J.; Verseyden, C.; Pluim,
D.; Jansen, L. E. T.; Bjornsti, M. A.; Schellens, J. H. M. J. Biol. Chem.
2004, 279, 54502. (f) Deng, C.; Brown, J. A.; You, D.; Brown, J. M.
Genetics 2005, 170, 591. (g) Kuo, C.-C.; Liu, J.-F.; Chang, J.-Y. J.
Pharmacol. Exp. Ther. 2006, 316, 946.
97% yield. Following the procedure of Lavergne et al.,15 we
subjected the acetylated amine to Vilsmeier conditions to
(14) (a) Tanizawa, A.; Kohn, K. W.; Kohlhagen, G.; Leteurtre, F.;
Pommier, Y. Biochemistry 1995, 34 7200. (b) Wang, X.; Wang, L.-K.;
Kingsbury, W. D.; Johnson, R. K.; Hecht, S. M. Biochemistry 1998, 37,
9399. (c) Wang, X.; Zhou, X.; Hecht, S. M. Biochemistry 1999, 38, 4374.
3514
Org. Lett., Vol. 8, No. 16, 2006

Figure 2. Salt-induced dissociation of the CPT-DNA-topoisomerase I ternary complex. Autoradiogram of the sequencing gel showing
the religation of the cleavage complex after addition of NaCl to a concentration of 0.35 M. At the times listed, the reactions were stopped
by the addition of SDS and proteinase K. The reaction shown in lanes 1-8 was carried out in the presence of 50 µM 1. Lanes 9-16 were
in the presence of 50 µM 5. Lanes 17-24 were in the presence of 50 µM 4. Lane 25 is a Maxam-Gilbert G-lane.
afford aldehyde 7 in 58% yield. Reduction of 7 to alcohol 8
with NaBH4 in EtOH proceeded nearly quantitatively.
Bromination of 8 in toluene with PBr3 at reflux for 18 h
gave a mixture of mono- and dibromides, which were
subjected to bromination conditions again to give key
intermediate 9 in 67% yield.
Pyrimidone 10 was prepared in eight steps from the
commercially available 2,4-dichloropyrimidine according to
a published procedure (Scheme 2).11,16 Pyrimidone 10 was
then condensed with 9 using tBuOK in DME at reflux to
afford key intermediate 11 in 69% yield. Radical-mediated
cyclization of 11 using tris(trimethylsilyl)silane and AIBN
as the radical initiator in benzene at reflux afforded 10,11-
MDO-14-azaCPT (5) as a colorless solid in 6% yield after
purification by column chromatography.
The cytotoxic effects of the analogues tested confirm this
trend. The IC50 values toward A549 cells were determined
using an MTT assay, as shown in Table 1. Compound 4 was
found to have an IC50 value >10 µM, whereas CPT itself
had an IC50 value of 0.15 µM. The increased persistence of
the ternary complex resulting from the introduction of the
10,11-MDO group in 5 resulted in an IC50 value of 1.28 µM.
Finally, the ability of CPT and 10,11-MDO-14-azaCPT
to inhibit the relaxation16 of supercoiled plasmid DNA by
topoisomerase I was compared (Figure 3). These results
As shown in Figure 2, CPT analogue 5 possessed potency
comparable to that of CPT and produced stabilization at the
same sites in DNA. The salt-induced dissociation of the
CPT-DNA-topoisomerase I ternary complex revealed that
incorporation of the 10,11-MDO group actually did lead to
an increased persistence of the formed complex (Table 1).
Table 1. Topoisomerase I-Dependent Cytotoxicity of 1, 4, and
5 and First-Order Rate Constants for Their Dissociation from
the CPT-Topo I-DNA Ternary Complex
Figure 3. Plasmid relaxation assay comparison of 1 and 5 (80
µM) against wild-type (WT) topoisomerase I and mutants D533A
and D533E.
a
compound
rate constant k (×10-3 s-1
)
IC50 (µM)
1
4
5
18.3
115
21.5
0.15
>10
1.28
indicate that the two were comparable, although 5 possessed
a slightly weaker ability to inhibit topoisomerase I-mediated
a Determined using 50 µM 1, 4, and 5 at cleavage site 2.
(15) Lavergne, O.; Lesueur-Ginot, L.; Rodas, F. P.; Kasprzyk, P. G.;
Pommier, J.; Demarquay, D.; Pre’vost, G.; Ulibarri, G.; Rolland, A.;
Schiano-Liberatore, A.; Harnett, J.; Pons, D.; Camara, J.; Bigg, D. C. H. J.
Med. Chem. 1998, 41, 5410.
(16) Experimental details and full characterization of all intermediates
are available in the Supporting Information.
Specifically, the dissociation rate constant increased to 115
from 18.3 when the C14-H group was replaced with N.
When the 10,11-MDO group was additionally incorporated,
the rate constant returned to 21.5, similar to that of CPT.
Org. Lett., Vol. 8, No. 16, 2006
3515

DNA relaxation. A direct comparison of the time course of
relaxation in the presence of 500 µM 1 and 5 gave similar
results, albeit with slightly greater inhibition in the presence
of 5 (Figure 4). More important, however, was the compari-
interactions with the C20 hydroxyl group of CPT and thus
is a useful site to probe. Removal of the carboxylate
altogether (D533A) and introduction of glutamic acid in lieu
of aspartic acid (D533E) still afforded topoisomerases I able
to relax supercoiled plasmid DNA (lanes 5 and 8). However,
both mutations prevented CPT from inhibiting this relaxation,
as shown in lanes 6 and 9. In a similar manner, 5 also no
longer inhibits relaxation (lanes 7 and 10) which is consistent
with the thesis that both CPT and 10,11-MDO-14-azaCPT
bind in the same manner to the DNA-topoisomerase I binary
complex.
These results confirm the hypothesis that appropriate
structural changes in CPT distant from the mechanistically
critical D and E rings can correct detrimental changes
accompanying modification of the D/E rings. Specifically,
it is possible to modulate the off-rate resulting from introduc-
tion of an aza group into the 14-position of CPT by
introducing additional structural features far from the 14-
position. Thus, 10,11-MDO-14-azaCPT represents another
important step toward the development of a CPT analogue
with useful therapeutic properties.
Figure 4. Kinetics of the relaxation of supercoiled pSP64 plasmid
DNA in the presence of CPT and 10,11-MDO-14-azaCPT. The
supercoiled DNA was incubated at 37 °C with 0.05 ng of human
topoisomerase I alone (diamonds) or in the presence of 500 µM
CPT (squares) or 500 µM 10,11-MDO-14-azaCPT (triangles).
Aliquots were treated with loading buffer containing 1% SDS at
predetermined times and analyzed by 1% agarose gel electrophore-
sis.
Acknowledgment. This work was supported by NIH
Research Grant 78415.
Supporting Information Available: Experimental pro-
cedures and full characterization for all compounds. Protocols
for the cytotoxicity assay. This material is available free of
charge via the Internet at http://pubs.acs.org.
son of the effects of CPTs 1 and 5 on topoisomerase I
mutants. Specifically, we prepared two modified topoi-
somerases I17 (D533A and D533E). Asp533, as indicated
above, is thought to be involved in hydrogen bonding
OL0611604
(17) Fiorani, P.; Bjornsti, M. Ann. N.Y. Acad. Sci. 2000, 922, 65.
3516
Org. Lett., Vol. 8, No. 16, 2006