Synthesis and anticancer activities of ageladine A and structural analogs

Article abstract of DOI:10.1016/j.bmcl.2009.11.036

A series of ageladine A analogs that include 2-aminoimidazo[4,5-c]azepines (seven-membered rings) and 2-amino-3H-imidazo[4,5-c]pyridine (six-membered rings) derivatives were synthesized and evaluated for their anticancer effects against several human cancer cell lines and MMP-2 inhibition in vitro. Only compounds possessing the aromatic azepine (seven-membered ring) core showed anticancer activity with IC₅₀ values in the low micromolar range.

Full text of DOI:10.1016/j.bmcl.2009.11.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 83–86
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer activities of ageladine A and structural analogs
*
Yuelong Ma, Sangkil Nam, Richard Jove, Kenichi Yakushijin, David A. Horne
Department of Molecular Medicine, Beckman Research Institute at City of Hope, Duarte, CA 91010, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ageladine A analogs that include 2-aminoimidazo[4,5-c]azepines (seven-membered rings) and
2-amino-3H-imidazo[4,5-c]pyridine (six-membered rings) derivatives were synthesized and evaluated
for their anticancer effects against several human cancer cell lines and MMP-2 inhibition in vitro. Only
compounds possessing the aromatic azepine (seven-membered ring) core showed anticancer activity
with IC50 values in the low micromolar range.
Received 17 August 2009
Revised 8 November 2009
Accepted 10 November 2009
Available online 14 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anticancer
Ageladine A
2-Aminoimidazole
Oroidin
Pictet–Spengler
N
N
N
Ageladine A (1a) is a unique imidazole–pyrrole-based marine
natural product which was isolated in 2003 from the sponge Agelas
nakamurai (Fig. 1).1 Bioactivity investigations determined that 1a is
an inhibitor of matrix metalloproteinases (MMPs). Its use as a
potential pH sensitive membrane permeable dye has also been
described.2 Several syntheses of ageladine A and related analogs
have been published in recent years.3–8 In an effort to discover
new anticancer lead structures for drug development, a collection
of agelidine A-related compounds was synthesized and tested
against different human cancer cell lines. Herein, we report the
results of this study and a novel class of anticancer fused seven-
membered ring 2-aminoimidazole azepine heterocycles 2.
In 1994, we disclosed that 2-aminoimidazoles such as 3 under-
go a facile Pictet–Spengler reaction with aldehydes to yield
2-aminoimidazoazepines 4 in excellent yields (Scheme 1).9 This
key reaction enables the construction of the bicyclic core of agela-
dine A and was independently adapted by Karuso in 2004 for the
synthesis of the natural product.8 Due to the important biological
activities reported for ageladine A, we recently decided to pursue
this reaction in greater depth and extend the methodology for
the preparation and biological evaluation of ageladine A analogs.
Of the 12 initial analogs prepared, eight of them are 3H-imi-
dazo[4,5-c]pyridin-2-amine (six-membered ring) analogs that in-
H2N
H2N
N
N
H
N
R
NH
Br
Br
ageladine A (1a)
2
Figure 1. Structure of ageladine A and compound 2.
R
O
N
NH2
n
H
N
n
NH
H2N
H2N
N
H
Pictet-Spengler
cyclization
N
H
R
3
4
n = 2
n = 2
Scheme 1. Pictet–Spengler cyclization of 2-aminoimidazoles.
N
N
N
H2N
H2N
N
N
H
N
R
R
1a-h
2e-h
clude the natural product, ageladine
A (1a) and four are
imidazo[4,5-c]azepin-2-amine (seven-membered ring) derivatives
comprising 2 (Fig. 2).
a
b
NH
c
g
R =
NH
Br
R =
R =
R =
R =
d
h
R =
R =
N
H
Br
e
R =
f
Cl
* Corresponding author.
E-mail address: DHorne@coh.org (D.A. Horne).
Figure 2. Ageladine A and analogs.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.036

84
Y. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 83–86
These analogs were prepared using Pictet–Spengler methodol-
N
N
ii
i
6a'-c', d
H2N
H2N
ogy followed by oxidation to the requisite aromatic system.
Starting from readily available 2-amino-4-(3-aminopropyl)-1H-
imidazole (3) and 2-aminohistamine (5),10,11 the corresponding
Pictet–Spengler cyclization products 412 and 6 were produced in
good yields from reaction with aldehyde substrates 7 (Scheme 2).
With tetrahydro ring products 4 and 6 in hand, oxidation to the
corresponding aromatic system was investigated. This was not as
straightforward as anticipated. While Karuso describes the use of
chloranil for the synthesis of ageladine A, the use of this oxidant
could not be generally applied to the azepine series. For instance,
attempts to use chloranil in oxidizing pyrrole–azepines 4, no aro-
matic products corresponding to 2 could be isolated. After experi-
menting with several different reagents and reaction conditions,
moderate success was achieved from a two-step procedure utiliz-
ing elemental bromine in methanesulfonic acid followed by treat-
ment with potassium tert-butoxide in air (Scheme 3).
N
N
N
H
N
H
R
R
1a'-c', 1d
1a,b,c
a'
a
NBoc c'
d
R =
R =
NBoc b' R =
R =
R =
N
Br
CO2Et
Br
NH
c
R =
NH
Br
b R =
N
H
Br
Scheme 4. Reagents and conditions: (i) chloranil (3 equiv), CHCl3, 80 °C, 16 h, 10
(50%), 1b0 (10%), 1c0 (15%), 1d (13%); (ii) TFA/CH2Cl2, 1a (95%), 1b (95%) or K2CO3,
MeOH, 1c (80%).
Initial oxidation with bromine at elevated temperatures
(110 °C, sealed tube) produced a mixture of products which pri-
marily consisted of dehydro intermediate 8.13 This intermediate
can be isolated and characterized. Only small amounts of the de-
sired aromatic system 214 were obtained under these conditions.
Upon exposure of 8 to potassium tert-butoxide in air, modest but
sufficient amounts of 2 were produced and used for bioactivity
studies.
The preparation of ageladine A (1a) and analogs 1b–d was
achieved by treatment of precursor 6 with chloranil which afforded
the desired aromatic derivatives after deprotection with either tri-
fluoroacetic acid or potassium carbonate (Scheme 4).
N
i
6e-h
H2N
N
N
H
R
1e-h
e
g
h
R =
R =
f
R =
R =
Cl
Scheme 5. Reagents and conditions: (i) Br2 (2 equiv), MeSO3H, 110 °C, 16 h, 1e
(32%), 1f (30%), 1g (31%), 1h (30%).
Finally, oxidation of 6e–f was best achieved using bromine in
methanesulfonic acid to afford the desired ageladine A analogs
1e–f (Scheme 5).
Next, biological assays were performed to determine the effects
of these analogs on cancer cell viability and MMP-2 activity. All
derivatives were screened for anticancer activities against human
R
O
N
NH2
n
7a'-h
H
N
n
NH
H2N
DU145
H2N
N
H
Pictet-Spengler
cyclization
N
H
DMSO 1 uM
5 uM 10 uM
R
140
120
100
80
3
4e-h n = 2
6a'-h n = 1
n = 2
5 n = 1
60
40
NBoc
a'
e
b'
f
c'
g
R =
NBoc
Br
R =
R =
d
R =
R =
R =
R =
N
Br
CO2Et
20
0
h
R =
2e
2f
2g
2h
Cl
Compd. #
Scheme 2. Reagents and conditions: 3ꢀ2HCl, 7e–h, H2O/EtOH, Na2CO3, 1 d, 23 °C:
products 4e (86%), 4f (70%), 4g (85%), and 4h (60%). Compounds 5, 7a0 or 7b0,
Sc(OTf)3, EtOH, 23 °C, 1 d: products 6a0 (21%) or 6b0 (22%); 5, 7c0–h, EtOH, 23 °C, 1 d:
products 6c0 (32%), 6d (40%), 6e (63%), 6f (51%), 6g (36%), 6h (38%).
DU145
DMSO 1 uM 10 uM
160
140
120
100
80
60
40
20
0
N
i
2e-h
+
4e-h
H2N
N
H
N
R
ii
8e-h
1a
1b
1c
1d
1e
1f
1g
1h
e
g
R =
f
R =
R =
h R =
Compd. #
Cl
Figure 3. Seven-membered ring aromatic analogs inhibit viabilities of DU145 cells
(A) but not six-membered analogs (B). Cells were treated with compounds at
various concentrations for 48 h. Cell viability was determined by an MTS assay
using manufacturer’s (Promega, Madiason, WI) protocol. Values are mean values of
three determinations with <10% deviation from the mean value.
Scheme 3. Reagents and conditions: (i) Br2 (2 equiv), MeSO3H, 110 °C, 16 h:
products 8e (25%) and 2e (11%), 8f (30%) and 2f (8%), 8g (33%) and 2g (6%), 8h (41%)
and 2h (5%); (ii) t-BuOK (1 equiv), THF, 16 h: products 2e (26%), 2f (47%), 2g (37%),
and 2h (25%).

Y. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 83–86
85
Table 2
DU145 prostate cancer cells (Fig. 3). Aromatic azepines 2e–h dis-
MMP-2 inhibitory activity of compounds 1a–h and 2e–fa
palyed anticancer activities in a dose-dependent manner. More-
over, the aromatic 2-aminoimidazo[4,5-c]azepine core plays an
important role in the anticancer activity since neither the non-aro-
matic seven-membered azepine derivatives (data not shown) nor
the six-membered pyridine (ageladine A) series showed activities.
Analogs of 1 and 2 were further tested with A2058 melanoma
and MDA-MB-435 breast cancer cell lines. Similarly, the seven-
membered ring aromatic compounds inhibited cell viabilities as
shown in Table 1.
Compds
% Inhibition @ 5
lM
% Inhibition @ 50 lM
1b
1c
1d
1e
1f
1g
1h
2e
2f
N/Ab
25
20
25
31
N/Ab
26
N/Ab
17
22
9
40
N/Ab
28
N/Ab
52
27
60
2g
2h
23
27
53
54
Table 1
Inhibition of cell viability by compounds 2e–ha
a
MMP-2 inhibition assay was performed using manufacturer’s (Enzo Life Sci-
ences AK-409) protocol. Values are mean values of two determinations with <10%
deviation from the mean value.
Compds
IC50
(lM) DU145
IC50
(l
M) A2058
IC50 (lM) MDA-MB-435
2e
2f
2g
2h
7.5
4.3
5
9.5
4.1
4.5
5.1
6.7
2.9
3.4
3.6
b
Strong fluorescence of the compound interfered with the assay measurement.
6.2
a
IC50 values were determined with an MTS assay after 48 h treatment. Values are
In addition, compounds 2e–h substantially reduced viabilities
of human multiple myeloma cells, including 8226, KMS11 and
U266 (Fig. 4).
mean values of three determinations with <10% deviation from the mean value.
Previous studies have reported that ageladine A (1a) inhibits
MMP-2 activities, which are involved in tumor angiogenesis. Con-
sistently, we observed potent inhibition of MMP-2 activity by agel-
8226 Multiple Myeloma
DMSO 1 uM
10 uM
adine A (1a) (IC50 = 1.7 0.2 lM) among all derivatives (1a–h and
140
120
100
80
2e–h). All other compounds showed only weak inhibition of MMP-
2 activities at higher concentrations (Table 2).
In conclusion, six- and seven-membered ring ageladine A ana-
logs have been synthesized via a two-step process that centers on
a Pictet–Spengler cyclocondensation followed by oxidative aro-
matization. The choice of oxidant and reaction conditions is
highly dependent on the desired ring system. While these two
classes of heterocycles have structural similarities, the bioactivi-
ties of the two systems are dramatically different. The seven-
membered analogs 2e–h represent a new pharmacophore that
possesses in vitro anticancer activity. This is contrast to the six-
membered pyridine series found in ageladine A. Further studies
expanding the medicinal chemistry of these derivatives along
with investigations directed at the biological mechanism of action
are under investigation.
60
40
20
0
2e
2e
2e
2f
2g
2h
2h
2h
Compd. #
KMS11
DMSO
1 uM
10 uM
160
140
120
100
80
60
40
20
0
Acknowledgments
The National Institutes of Health R01GM071985 and Chugai
Pharmaceutical Co. are gratefully acknowledged for support of this
work. We thank the Irell & Manella Graduate School of Biological
Sciences at City of Hope for a merit fellowship to Y.M.
2f
2g
Compd. #
U266 Multiple Myeloma
References and notes
DMSO
1 uM
10 uM
120
100
80
60
40
20
0
1. Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.; Yamashita, J.; Van Soest,
R. W.; Fusetani, N. J. Am. Chem. Soc. 2003, 125, 15700.
2. Bickmeyer, U.; Grube, A.; Klings, K. W.; Kock, M. Biochem. Biophy. Res. Commun.
2008, 373, 419.
3. Ando, N.; Terashima, S. Bioorg. Med. Chem. Lett. 2007, 17, 4495.
4. Meketa, M. L.; Weinreb, S. M. Org. Lett. 2006, 8, 1443.
5. Meketa, M. L.; Weinreb, S. M. Org. Lett. 2007, 9, 853.
6. Meketa, M. L.; Weinreb, S. M. Tetrahedron 2007, 63, 9112.
7. Meketa, M. L.; Weinreb, S. M.; Nakao, Y.; Fusetani, N. J. Org. Chem. 2007, 72,
4892.
8. Shengule, S. R.; Karuso, P. Org. Lett. 2006, 8, 4083.
9. Horne, D. A.; Yakushijin, K. PCT Int. Appl. (1994), W.O. Patent 9,424,136, 1994;
Chem. Abstr. 1994, 122, 81723. Horne, D. A.; Yakushijin, K.; U.S. Patent
5,834,609, 1998.
10. Lancini, G. C.; Arioli, V.; Lazzari, E.; Bellani, P. J. Med. Chem. 1969, 12,
775.
2f
2g
Compd. #
Figure 4. Compounds 2e–h inhibit viabilities of multiple myeloma cells. Cells were
treated with compounds at 1 M or 10 M for 48 h. Cell viability was determined
by an MTS assay. Values are mean values of three determinations with <10%
deviation from the mean value.
l
l
11. Lancini, G. C.; Lazzari, E. J. Heterocycl. Chem. 1966, 3, 152.

86
Y. Ma et al. / Bioorg. Med. Chem. Lett. 20 (2010) 83–86
12. Compound 4eꢀ2HCl: 1H NMR (DMSO-d6, 400 MHz) d 12 .31 (d, J = 17.0 Hz, 2H),
9.92 (br, 1H), 9.33 (br, 1H), 7.49 (s, 2H), 4.04 (d, J = 9.4 Hz, 1H), 3.38 (br, 2H),
2.67 (br, 2H), 2.26 (m, 1H), 1.92 (br, 2H), 1.02 (d, J = 6.5 Hz, 3H), 0.88 (d,
J = 6.5 Hz, 3H), 13C NMR (DMSO-d6, 100 MHz) d 146.8, 126.1, 124.6, 60.8, 44.4,
29.4, 26.3, 23.3, 19.1, 18.1.
NMR (CD3OD, 100 MHz)
33.3, 19.8.
d 167.2, 155.7, 143.6, 130.3, 124.3, 119.4, 47.5,
14. Compound 2e: 1H NMR (CDCl3, 400 MHz) d 8.79 (d, J = 6.8 Hz, 1H), 8.02 (d,
J = 10.0 Hz, 1H), 7.39 (dd, J = 10.0 Hz, J = 6.8 Hz, 1H), 7.29 (s, 2H), 4.24 (m, 1H),
1.34 (d, J = 6.8 Hz, 6H), 13C NMR (CDCl3, 100 MHz) d 174.0, 164.0, 163.8, 156.9,
149.8, 130.3, 127.3, 34.4, 22.2, HRMS(ESI) m/z calcd for C10H13N4 (M+H):
189.1140; found: 189.1141.
13. Compound 8e: 1H NMR (CD3OD, 400 MHz) d 6.73 (d, J = 9.4 Hz, 1H), 5.66
(m, 1H), 3.62 (d, J = 6.7 Hz, 2H), 3.21 (m, 1H), 1.12 (d, J = 6.8 Hz, 6H), 13C