Analogs of the marine alkaloid makaluvamines: Synthesis, topoisomerase II inhibition, and anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2007.02.065

Twelve analogs of makaluvamines have been synthesized. These compounds were evaluated for their ability to inhibit the enzyme topoisomerase II. Five compounds were shown to inhibit topoisomerase catalytic activity comparable to two known topoisomerase II

Full text of DOI:10.1016/j.bmcl.2007.02.065

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2890–2893
Analogs of the marine alkaloid makaluvamines: Synthesis,
topoisomerase II inhibition, and anticancer activity
Bidhan A. Shinkre,a Kevin P. Raisch,b Liming Fanb and Sadanandan E. Velua,*
aDepartment of Chemistry, University of Alabama at Birmingham, 901 14th Street South, Birmingham, AL 35294, USA
bDepartment of Radiation Oncology, University of Alabama at Birmingham, 1824 6th Ave South, Birmingham, AL 35294, USA
Received 18 December 2006; revised 16 February 2007; accepted 21 February 2007
Available online 25 February 2007
Abstract—Twelve analogs of makaluvamines have been synthesized. These compounds were evaluated for their ability to inhibit the
enzyme topoisomerase II. Five compounds were shown to inhibit topoisomerase catalytic activity comparable to two known topo-
isomerase II targeting control drugs, etoposide and m-AMSA. Their cytotoxicity against human colon cancer cell line HCT-116 and
human breast cancer cell lines MCF-7 and MDA-MB-468 has been evaluated. Four makaluvamine analogs exhibited better IC50
values against HCT-116 as compared to control drug etoposide. One analog exhibited better IC50 value against HCT-116 as com-
pared to m-AMSA. All 12 of the makaluvamine analogs exhibited better IC50 values against MCF-7 and MDA-MB-468 as com-
pared to etoposide as well as m-AMSA.
Ó 2007 Elsevier Ltd. All rights reserved.
For the past quarter of a century, global marine sources
have proven to be a rich source of a vast array of new
medicinally valuable compounds.1 These natural prod-
ucts exist as secondary metabolites in marine inverte-
brates such as sponges, bryazoa, tunicates, and
ascidians. As a result of the potential for new drug dis-
covery, marine natural products have attracted scientists
from different disciplines, such as organic chemistry,
bioorganic chemistry, pharmacology, and biology.
About a dozen of marine alkaloids are currently in var-
ious phases of human clinical trials for treatment of dif-
ferent cancers.2 The largest number of bioactive marine
alkaloids with novel structures has been isolated from
marine sponges.3,4 Sponges produce a plethora of chem-
ical compounds with widely varying carbon skeletons.
Most bioactive compounds from sponges have exhibited
a variety of activities such as anti-inflammatory, antitu-
mor, immunosuppressive, neurosuppressive, antiviral,
antimalarial, and antibiotic activities.4 While a number
of these alkaloids have been isolated in quantities suffi-
cient to ascertain their biological profile, many with un-
ique structures are available only in minute quantities,
precluding their thorough biological evaluations. Labo-
ratory synthesis of these alkaloids is the only practical
solution to this problem.
Marine sponges of the genera Latrunculia, Batzella,
Prianos, and Zyzzya are a rich source of alkaloids bearing
a pyrrolo[4,3,2-de]quinoline skeleton.5,6 This series of alka-
loids comprises of about 60 metabolites including discor-
habdins,7 epinardins,8 batzellines,9 isobatzellines,9
makaluvamines,10–15 and veiutamine.16 Pyrrolo[4,3,2-
de]quinoline alkaloids have shown a variety of biological
activities such as inhibition of topoisomerase I14 and II,10
cytotoxicity against different tumor cell lines,10,17 and anti-
fungal14 and antimicrobial activities.18 Pyrrolo[4,3,2-
de]quinoline alkaloids have recently received increasing
attention as a source of new anticancer drugs.19–24 Their
unique fused ring skeletons carrying interesting biological
properties have made them targets for several synthetic
and biological studies. There has been a rapid growth of
interest in the synthesis and biological evaluation of this
class of compounds and their analogs. Several reviews have
been published on the chemistry and bioactivity of this class
of compounds.6,25,26 Our interest is focused on makaluv-
amines which belong to this family of alkaloids. Makaluv-
amines A–P are a group of 16 marine alkaloids isolated
mainly from four species of marine sponges, namely the
Fijian sponge Zyzzyacf. marsailis,10 Indonesian sponge
Histodermella sp.,14 Pohnpeiansponge Zyzzya fuliginosa11,
and Jamaican sponge Smenospongia aurea15 (Fig. 1).
Keywords: Makaluvamine; Analog; HCT-116; MCF-7; MDA-MB-4-
68; Topoisomerase II.
*
Corresponding author. Tel.: +1 205 975 2478; fax: +1 205 934
2543; e-mail: svelu@uab.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.065

B. A. Shinkre et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2890–2893
Br
2891
HO
HN
O
HN
HN
O
HO
N
CH3
N
H
S
N
H
H2N
N
H
N
H
O
1, Makaluvamine A
2, Makaluvamine D
3, Makaluvamine F
Figure 1.
Makaluvamines have exhibited in vitro cytotoxicity
against human colon tumor cell line, HCT-116. The
cytotoxicities exhibited by makaluvamines against a
Chinese hamster ovary (CHO) cell line Xrs-6 have par-
alleled the data obtained with HCT-116.10 Makaluvam-
ines produce their anticancer activity by targeting the
enzyme topoisomerase II as shown by a decatenation
inhibition assay.10 According to a recent publication it
is possible that makaluvamines produce their anticancer
activity by other mechanisms as well.27
HN
O
HN
O
(6a-g)
NH2
R
N
Ts
CH3OH
N
Ts
R
N
H3CO
H
5
7a-g
HN
O
1. NaOCH3
CH3OH
2. TFA
N
R
N
4a-g
H
H
As a part of our research on marine natural product
analogs with potential pharmacological value, we have
been interested in studying the cytotoxicity of makaluv-
amine analogs. We propose to make these analogs by
introducing different substituents at the 7-position of
the pyrroloiminoquinone ring present in makaluvam-
ines. Many pyrroloiminoquinone alkaloids with proven
cytotoxicities were found to have substitutions at the
7-position of the ring. We decided to explore some
simple substitutions with increased steric bulk, hydro-
phobicity, and hydrophilicity at the 7-position of the
pyrroloiminoquinone ring and study their effects on
topoisomerase II inhibition and cytotoxicity. Proposed
target structures are given in Figure 2.
Scheme 1.
Treatment of the pyrroloiminoquinone derivative 5 with
different amine derivatives (6a–g) in anhydrous metha-
nol at room temperature provided the aminated com-
pounds 7a–g in 51–68% yield. Removal of tosyl
protecting group from the compounds 7a–g was accom-
plished by treatment with NaOMe in MeOH to obtain
the final products 4a–g in 48–62% yield. In the synthesis
of compounds 4a–b, intermediate products 7a–b were
not isolated as the amination and detosylation took
place in a single step leading to the formation of final
products. This type of detosylation in the presence of
amines was not unusual and has been previously
reported in the literature.29 All these compounds were
individually synthesized and fully characterized.
Makaluvamine analogs were synthesized from the pyr-
roloiminoquinone derivative 5. Synthesis of compound
5 has been reported in the literature by several groups.28
We prepared this compound following the 4,6,7-trim-
ethoxyindole approach described previously.29 This
compound was converted to the makaluvamine analogs
in two steps as outlined in Scheme 1.
Compounds 4a–g and 7c–g were evaluated for their
cytotoxicities against human breast cancer cell lines
MCF-7 and MDA-MB-468, and human colon cancer
cell line HCT-116. The dose of the compound that
inhibits 50% cell proliferation (IC50) was calculated
using the data generated from 2 to 4 independent tetra-
zolium-based (XTT) cytotoxicity assays (R&D Systems
Inc., Minneapolis, MN). Two known topoisomerase II
targeting drugs, m-AMSA and etoposide, were included
for comparisons. Results of cytotoxic assays are summa-
rized in Table 1. HCT-116 cells were shown to be the
most sensitive to etoposide and m-AMSA with IC50
doses of 1.7 and 0.7 lM, respectively. MDA-MB-468
cells showed IC50 doses of 13.6 and 8.5 lM for etoposide
and m-AMSA, respectively. MCF-7 cells were shown to
be the least sensitive with IC50 values of 35.6 and
21.7 lM for etoposide and m-AMSA, respectively. Sev-
eral of makaluvamine analogs have shown significantly
better inhibition than the control drugs in these assays.
Four makaluvamine analogs (4c, 7d, 7f, and 7g) exhib-
ited better IC50 values against HCT-116 as compared
to control drug etoposide. One analog (7d) exhibited
HN
N
H
R
N
H
O
4, Makaluvamine analogs
R =
CH3
(a)
CH2CH3
(b)
CH2
(c)
CH2 CH2
(d)
Br
Br
NH
CH2 CH2
(g)
CH2 CH2
(e)
OH
CH2 CH2
(f)
OH
Figure 2.

2892
B. A. Shinkre et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2890–2893
Table 1. In vitro cytotoxicity and inhibition of topoisomerase II catalytic activities of makaluvamine analogs
Structure
Compound R =
Inhibition of cancer cell
proliferation (IC50a, lM)
Topoisomerase II inhibitionb
HCT-116
MCF-7
MDA-MB-468
4a
4b
–CH3
–CH2CH3
2.5 1.3
3.6 0.7
1.3 0.1
1.3 0.3
0.4 0.05
0.4 0.03
À
CF3COO
–
HN
4c
4d
1.3 0.2
3.9 1.2
1.0 0.4
1.7 0.5
0.3 0.18
0.3 0.27
++
+
CH2
N
R
N
CH2 CH2
CH2 CH2
H
H
O
2
13.2 0.9
14.4 1.2
3.2 0.6
0.6 0.05
4.5 0.8
À
OH
OH
Br
4f
13.2 3.1
+++
CH2
CH2
Br
NH
CH2 CH2
4g
5.2 0.8
1.6 0.2
0.4 0.01
À
7c
7d
2.7 1.5
1.8 0.2
1.5 0.1
1.0 0.01
0.8 0.1
+++
CH2
CF3COO
HN
d
0.5 0.03
—
CH2 CH2
N
R
N
7e
7f
5.3 0.6
1.0 0.2
5.1 0.4
2.3 1.1
1.5 0.14
1.5 0.9
+++
CH2 CH2
OH
OH
H
O
S O
O
Br
d
—
CH2
CH2
CH3
Br
NH
CH2 CH2
d
7g
0.8 0.3
1.2 0.03
1.4 0.9
—
c
c
Etoposide
m-AMSA
—
—
1.7 0.2
0.7 0.3
35.6 3.4
21.7 2.5
13.6 0.6
8.5 1.2
+
++
a The dose that inhibits 50% cell proliferation (IC50) was determined in human colon cancer cell line, HCT-116, and the human breast cancer cell
lines, MCF-7 and MDA-MB-468 (ATCC, Manassas, VA). The IC50 doses from 2 to 4 independent XTT assays performed in triplicate were
combined for an average standard deviation.
b Topoisomerase II (4U) was incubated with 500 ng plasmid DNA containing vehicle (DMSO) or 100 lM drug compound as described in the Topo II
Drug Screening Kit protocol (TopoGEN, Inc). m-AMSA and etoposide were used as positive controls. Relaxed DNA was separated using non-
ethidium bromide (EtBr) agarose gels, then stained with EtBr and quantified using Kodak Gel Logic Imaging System and Molecular Imaging
software (Eastman Kodak Co., Rochester, NY). Inhibition of relaxation of plasmid DNA or catalytic activity is reported as: À none, + low, ++
moderate, or +++ strong.
c Not applicable.
d Not determined.
better IC50 value against HCT-116 as compared to
m-AMSA. All twelve of the makaluvamine analogs
exhibited better IC50 values against MCF-7 and
MDA-MB-468 as compared to etoposide as well as
m-AMSA. Compound that showed best activity against
HCT-116 is the N-tosyl-6-phenethylamino derivative
(7d) with an IC50 value of 0.5 lM. The compound that
showed best activity against MCF-7 is the benzyl amino
derivative (4c) with an IC50 value of 1.0 lM. Benzyl ami-
no derivative, 4c, and phenethyl amino derivative, 4d,
showed the best activity against MDA-MB-468 with
IC50 value of 0.3 lM for each.
The final products (4a–g) and the intermediate products
(7c–g) were also screened for their ability to inhibit
topoisomerase II enzymatic activity. Topoisomerase II
functions by generating a double-stranded DNA break
followed by resealing of the break. Topoisomerase II
inhibitors will interfere with the breakage-rejoining reac-
tion thereby trapping the enzyme in a cleavage complex.
In order to find out the ability of our compounds to in-
hibit topoisomerase II enzymatic activity, we used a
topoisomerase-II drug screening kit (TopoGEN, Inc.,
Port Orange, FL) that uses a supercoiled plasmid
DNA substrate (pRYG) which contains one topoiso-

B. A. Shinkre et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2890–2893
2893
5. Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1, and previous
reviews in the series.
6. Antunes, E. M.; Copp, B. R.; Davies-Coleman, M. T.;
Samaai, T. Nat. Prod. Rep. 2005, 22, 62.
7. Dijoux, M.-G.; Gamble, W. R.; Hallock, Y. F.; Cardel-
lina, J. H., II; Van Soest, R.; Boyd, M. R. J. Nat. Prod.
1999, 62, 636.
8. D’Ambrosio, M.; Guerriero, A.; Chiasera, G.; Pietra, F.
Tetrahedron 1996, 52, 8899.
9. Chang, L. C.; Otero-Quintero, S.; Hooper, J. N. A.;
Bewley, C. A. J. Nat. Prod. 2002, 65, 776.
10. Radisky, D. C.; Radisky, E. S.; Barrows, L. R.; Copp, B.
R.; Kramer, R. A.; Ireland, C. M. J. Am. Chem. Soc. 1993,
115, 1632.
11. Schmidt, E. W.; Harper, M. K.; Faulkner, D. J. J. Nat.
Prod. 1995, 58, 1861.
12. Venables, D. A.; Concepcion, G. P.; Matsumota, S. S.;
Barrows, L. R.; Ireland, C. M. J. Nat. Prod. 1997, 60,
408.
13. Casapullo, A.; Cutignano, A.; Bruno, I.; Bifulco, G.;
Debitus, C.; Gomez-Paloma, L.; Riccio, R. J. Nat. Prod.
2001, 64, 1354.
14. Carney, J. R.; Scheuer, P. J.; Kelly-Borges, M. Tetrahe-
dron 1993, 49, 8483.
15. Hu, J.-F.; Schetz, J. A.; Kelly, M.; Peng, J.-N.; Ang, K.
K. H.; Flotow, H.; Leong, C. Y.; Ng, S. B.; Buss, A.
D.; Wilkins, S. P.; Hamann, M. T. J. Nat. Prod. 2002,
65, 476.
16. Venables, D. A.; Barrows, L. R.; Lassota, P.; Ireland, C.
M. Tetrahedron Lett. 1997, 38, 721.
merase II cleavage/recognition site. Two topoisomerase
II inhibiting cancer drugs m-AMSA and etoposide were
used as controls in these assays. The results of our assays
are summarized in Table 1. Five out of nine tested mak-
aluvamine analogs exhibited inhibition of topoisomer-
ase II catalytic activity comparable to that of
etoposide and m-AMSA. Three of these compounds
(4f, 7c, and 7e) showed the strongest inhibition of cata-
lytic activity of topoisomerase II.
In conclusion, several analogs of makaluvamines have
been synthesized and characterized. Anticancer activity
of these analogs against human colon tumor cell line,
HCT-116, and human breast cancer cell lines, MCF-7
and MDA-MB-468, was evaluated. Four makaluvamine
analogs exhibited better IC50 values against HCT-116 as
compared to control drug etoposide. One analog exhib-
ited better IC50 value against HCT-116 as compared to
m-AMSA. All 12 of the makaluvamine analogs exhib-
ited better IC50 values against MCF-7 and MDA-MB-
468 as compared to etoposide as well as m-AMSA.
Makaluvamine analogs were also evaluated for their
ability to inhibit topoisomerase II enzymatic activity
and found that five makaluvamine analogs exhibited
inhibition of topoisomerase II comparable to etoposide
and m-AMSA.
17. Gunasekera, S. P.; Zuleta, I. A.; Longley, R. E.; Wright,
A. E.; Pomponi, S. A. J. Nat. Prod. 2003, 66, 1615.
18. Perry, N. B.; Blunt, J. W.; Munro, M. H. G. Tetrahedron
1988, 44, 1727.
19. Kokoshka, J. M.; Capson, T. L.; Holden, J. A.; Ireland, C.
M.; Barrows, L. R. Anti-Cancer Drugs 1996, 7, 758.
Acknowledgments
Authors wish to acknowledge the financial support by
Breast Spore pilot grant from the University of Ala-
bama at Birmingham (UAB). SEV also wishes to
acknowledge the financial support by a faculty develop-
ment grant from UAB (FGDP).
´ ´
´
20. Beneteau, V.; Pierre , A.; Pfeiffer, B.; Renard, P.; Besson,
T. Bioorg. Med. Chem. Lett. 2000, 10, 2231.
´ ´
21. Beneteau, V.; Besson, T. Tetrahedron Lett. 2001, 42, 2673.
22. Zhao, R.; Oreski, B.; Lown, W. Bioorg. Med. Chem. Lett.
1996, 6, 2169.
Supplementary data
23. Legentil, L.; Lesur, B.; Delfourne, E. Bioorg. Med. Chem.
Lett. 2006, 16, 427.
24. Legentil, L.; Benel, L.; Bertrand, V.; Lesur, B.; Delfourne,
E. J. Med. Chem. 2006, 49, 2979.
25. Urban, S.; Hickford, S. J. H.; Blunt, J. W.; Munro, M. H.
G. Curr. Org. Chem. 2000, 4, 778.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.02.065.
26. Ding, Q.; Chichak, K.; Lown, J. W. Curr. Med. Chem.
1999, 6, 1.
References and notes
27. Dijoux, M-G.; Schnabel, P. C.; Hallock, Y. F.; Boswell, J.
L.; Johnson, T. R.; Wilson, J. A.; Ireland, C. M.; Soest,
R.; Boyd, M. R.; Barrows, L. R.; Cardellina, J. H., II
Bioorg. Med. Chem. 2005, 13, 6035.
28. Harayama, Y.; Kita, Y. Curr. Org. Chem. 2005, 9,
1567.
29. Sadanandan, E. V.; Pillai, S. K.; Lakshmikantham, M. V.;
Billimoria, A. D.; Culpepper, J. S.; Cava, M. P. J. Org.
Chem. 1995, 60, 1800.
1. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote,
P. T.; Prinsep, M. R. Nat. Prod. Rep. 2005, 22, 15.
2. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2004, 67, 1216.
3. Sipkema, D.; Franssen, M. C. R.; Osinga, R.; Tramper, J.;
Wijffels, R. H. Mar. Biotechnol. 2005, 7, 142.
4. Higa, T.; Tanaka, J.; Kitamura, A.; Koyama, T.; Takah-
ashi, M.; Uchida, T. Pure Appl. Chem. 1994, 66, 2227.