The first total synthesis and biological evaluation of marine natural products ma'edamines A and B

Article abstract of DOI:10.1016/j.bmcl.2012.12.033

We have developed the first total syntheses of marine natural products ma'edamines A (18) and B (20). Structurally, they contain a pyrazine-2-(1H)-one core and were screened for antiproliferative activity on several cancer cell lines. Out of the six cell lines tested, ma'edamines A and B showed significant cytotoxicity against human colon cancer line COLO 205 (IC₅₀ 7.9 and 10.3 μM, respectively), breast cancer cell line MCF-7 (IC₅₀: 6.9 and 10.5 μM, respectively) and human lung adenocarcinoma cell line A549 (IC₅₀: 12.2 and 15.4 μM, respectively). The apoptotic effect of ma'edamines was confirmed by comet assay. Hence ma'edamines are likely to be useful as leads for development of a new class of anti-cancer agents.

Full text of DOI:10.1016/j.bmcl.2012.12.033

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The first total synthesis and biological evaluation of marine natural
products ma’edamines A and B
Sanjay Saha a,b, Ch. Venkata Ramana Reddy b, T. Chiranjeevi c, Uma Addepally c, T. S. Chinta Rao a,b
,
Balaram Patro a,
⇑
a GVK Biosciences Private Limited, Medicinal Chemistry Division, 28A, IDA Nacharam, Hyderabad 500076, India
b Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500085, India
c Center for Innovative Research, IST, JNT University, Kukatpally, Hyderabad, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed the first total syntheses of marine natural products ma’edamines A (18) and B (20).
Structurally, they contain a pyrazine-2-(1H)-one core and were screened for antiproliferative activity on
several cancer cell lines. Out of the six cell lines tested, ma’edamines A and B showed significant cytotox-
Received 19 September 2012
Revised 7 December 2012
Accepted 11 December 2012
Available online xxxx
icity against human colon cancer line COLO 205 (IC50 7.9 and 10.3
line MCF-7 (IC50: 6.9 and 10.5 M, respectively) and human lung adenocarcinoma cell line A549 (IC50
12.2 and 15.4 M, respectively). The apoptotic effect of ma’edamines was confirmed by comet assay.
lM, respectively), breast cancer cell
l
:
l
Keywords:
Ma’edamine A
Cytotoxic
Hence ma’edamines are likely to be useful as leads for development of a new class of anti-cancer agents.
Ó 2012 Published by Elsevier Ltd.
Antiproliferative
Comet assay
HER2
The marine ecosystem is a treasure of diverse compounds and a
rich source of biological diversity which translates into excellent
chemical diversity.1–3 Many potent compounds of marine origin
or their analogues have entered clinical trials or have been ap-
proved as drugs.4 Selected examples include: bryostatin 1, dolast-
atin 10, discodermolide, bengamides and Ecteinascidine 743.5–9
Ma’edamines A (18) and B (20) are cytotoxic compounds, isolated
from the extract of the Okinawan marine sponge Suberea sp.,
We report herein the syntheses of ma’edamines A and B, and
results of their biological evaluation; which includes assessment
of anti-proliferative activity on several cancer cell lines. The
ma’edamines were found to be moderately potent in vitro (low
micromolar IC50) and induced apoptosis, which was confirmed by
comet assay.15
We envisioned the synthesis of ma’edamines by two routes
shown below in the retro synthesis (Fig. 2).
exhibiting ErbB2 kinase inhibitory activity (IC50: 6.7
l
g/mL).10
Both routes revealed an
building block, the second component being an
tive F2 in the former and an -amino acid F4 in the latter. The
a-amino aryl ketone F3 as the common
They belong to a family of marine bromotyrosine alkaloids,11 con-
taining a unique pyrazine-2-(1H)-one core structure. Compounds
containing this scaffold are not known in literature for exhibiting
anti-cancer activity, whereas many selective ErbB2 (human epider-
mal growth factor receptor or HER2) kinase inhibitors, or HER2/
EGFR dual inhibitors bearing a 4-amino-quinazoline, 4-aminoquin-
oline are approved as drugs (Fig. 1).12 Due to the involvement of
the EGFR family of tyrosine kinases in the development of several
types of solid tumors like non small cell lung cancer, breast cancer,
head and neck cancer, etc., they continue to be the subject of
further development through rational design.13 Recent examples
include development of pyrollo[3,2-d]pyrimidine and pyrimidine
scaffolds as dual EGFR/HER2 inhibitors.14
a-keto acid deriva-
a
a
-keto acid derivative F2 (10) was synthesized from p-anisaldehyde
in three steps by a reported procedure.16 The second building block
8 was readily synthesized from 4-hydroxyacetophenone via bromi-
nations, followed by a Delépine reaction of the resulting bromoaryl
ketone 417 (Scheme 1). Compound 9 was also prepared by the same
route.
The acid–amine coupling of the key intermediates 8 and 10 by a
standard protocol using TBTU/n-methylmorpholine/DMF afforded
intermediate 11 (Scheme 2). Ring closure using ammonium acetate
in ethanol yielded pyrazinone derivative 12. Alkylation of 12 with
1,3-dibromopropane,18 furnished a mixture of mono and bis-
alkylated products, which was difficult to purify. Alkylation of 12
with (3-chloropropyl)-dimethyl-amine 14, was not regiospecific
and also resulted in formation of a complex mixture. Therefore,
chloropyrazine derivative 13 was prepared by reaction of
⇑
Corresponding author. Tel.: +91 40 66281666; fax: +91 40 66281505.
E-mail address: balaram.patro@gvkbio.com (B. Patro).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.12.033
Please cite this article in press as: Saha, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.033

2
S. Saha et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
F
O
O
OH
O
Br
Br
+
a
N
H
OH
HN
O
HN
N
Cl
H
N
OH
OH
O
O
MeO
MeO
N
MeO
O
O
N
N
N
51%
Br
Br
Br
Br
O
11
N
O
OH
Br
8
NH3
10
Geftinib
Erlotinib
O
Cl
O
F
O
Br
Br
S
H
N
Cl
N
MeO
O
NH
HN
b
c
Br
OH
Br
O
N
N
Lapatinib
42%
64%
13
12
N
OH
Br
Br
Figure 1. FDA approved tyrosine kinase inhibitors.
Br
f
d
e
MeO
Cl
N
18: Ma'edamine A; R=Me, 25%
20: Ma'edamine A; R=Me, 24%
Br
Br
Br
N
H
N
Route A
Br
alkyl
MeO
MeO
O
MeO
O
OH
O
16: R=NMe2, 45%
halide: Cl
R
O
R
17: R=NHMe, <5%
19: R= Br
14: R= NMe2
15: R=NHMe
Br
N
F1
Br
F2
+
OH
Route B
O
Scheme 2. Reagents and conditions: (a) TBTU/NMM/DMF, rt, 2 h; (b) NH4OAc,
EtOH; (c) POCl3/DMF; (d) alkyl halide, base; (e) MeNH2 (MeOH), rt; (f) dil AcOH,
160 °C, 16 h.
Br
+
H2N
O
Br
OH
OH
NH
P
F3
Br
F4
O
OR2
R1
R
O
R1
O
N
H
Br
+
Br
Figure 2. Retro synthetic approach for synthesis of pyrazin-2(1H)-ones showing
key fragments (F1–F4).
a
NH
Boc
N
OH
H
22
NH3+Br-
Br
Br
O
23, R=Boc
23a, R=H
OR2
pyrazinone 12 with phosphoryl chloride. O-Alkylation of 13 with
(3-chloropropyl) dimethyl-amine 14 afforded 16. The best yields
were obtained when the alkylation was done in butanone, in
presence of cesium carbonate as base and one equivalent of
sodium iodide added as an additive. Treating the chloropyrazine
derivative 16 with 90% acetic acid and water at 160 °C for
12–16 h in a sealed tube afforded ma’edamine A 18.
The alkylation of chloropyrazine derivative 13 with (3-chloropropyl)-
methyl-amine 15 was very low yielding (<5%). Therefore 13 was
alkylated with 1,3-dibromopropane and the resulting product
was treated with dimethylamine to afford 17. Similar de-protection
of 17 yielded ma’edamine B in a modest yield. Both 18 and 20 were
characterized by 1H NMR, 13C NMR and HR-MS analyses and the
analytical data was consistent with the literature.10 The structure
of ma’edamine A was further confirmed by NOESY, HMBS and HSQC
NMR analyses.
Br
9
R1=
R2=
H
Br
MeO
b
H
N
O
N
O
c
Br
O
R1
Br
R1
24
N
N
NR3R4
OR2
Br
18: R3=R4=Me
Br
20: R3=H, R4=Me
Scheme 3. Reagents and conditions: (a) HATU, TEA, DMF, 4 h; (b) 4(M) HCl in
1,4-dioxane, rt, 4 h; (c) amine in THF, rt, 16–24 h.
coupling protocol. Treatment of the amide 23, with hydrochloric
acid (4 M in dioxane) afforded a mixture of 23a and 24. We found
that treatment of this mixture with anhydrous dimethylamine in
methanol resulted in cyclization of 23a to 24 followed by air oxida-
tion to afford ma’edamine A 18. Ma’edamine B was obtained sim-
ilarly by treating the mixture of 23a and 24 with methylamine in
THF. These compounds were characterized by 1H NMR and mass
spectra which were identical to the ones prepared by Scheme 2.
Ma’edamines A and B were studied for their antiproliferative activ-
ity by MTT assay,19 trypan blue staining20 and apoptotic potential
by comet assay15,21 as well as DNA laddering.22
In order to expand the scope of our synthetic method for creat-
ing small focused library of analogues we have also developed a
second route using different building blocks. The second approach
of synthesis of ma’edamines is depicted in Scheme 3. N-Boc
a-amino
acid 22 and
a
-amino aryl ketone 9 were coupled by a standard
OH
a
R
R
R
Cells in the exponential phase of growth were exposed to
the test substance. The duration of exposure was usually set as
the time required for minimal damage to occur. After addition of
the drug, the cells were allowed to proliferate for two to three pop-
ulations doubling times (PDT’s) in order to distinguish between
cells that remain viable and are capable of proliferation and those
that cannot proliferate. The number of surviving cells were then
determined indirectly by reduction of MTT ((3,4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) by the mitochondrial
dehydrogenase of live cells to a purple formazan product.19 The
amount of MTT–formazan produced was determined spectrophoto-
metrically at 570 nm after dissolving it in DMSO.
Br
Br
Br
Br
Br
d
Br
Br
N
c
N
N
48%
O
N
O
O
O
1
Br
2
4, 5
6, 7
b
3
R
Br
Br
e
2, 3, 6, 8; R= OH;
3, 5, 7, 9; R= OCH2CH2CH2Br
O
59% for 2 steps
Br
8, 9
NH3
The cytotoxicity data is expressed as IC50 values in lM (Table 1).
Scheme 1. Reagents and conditions: (a) Br2, AcOH, rt, 2 h; (b) BrCH2CH2CH2Br,
K2CO3, DMF, rt, 18 h; (c) Br2, CHCl3, rt, 2 h; (d) Hexamethylene tetramine, CHCl3, rt,
18 h; (e) MeOH, HBr (aq), 48 h, 59% for two steps.
Etoposide, a well-known anti-cancer drug was used as the positive
control for comparison.23 Ma’edamines and intermediates were
Please cite this article in press as: Saha, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.033

S. Saha et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Table 1
Antiproliferative activity of ma’edamines and its intermediates
Compound
Structure
IC50
A-549
(lM)
COLO-25
7.9
MCF-7
6.9
THP-1
B16-F10
16.7
HepG2
29.6
Br
Br
O
N
18
16
12
12.2
14.4
52
46.2
NMe2
NMe2
MeO
O
Br
N
H
Br
Br
Br
O
N
N
nt
nt
nt
nt
87.3
63.2
146
nt
nt
MeO
Cl
Br
N
Br
OH
Br
124.7
MeO
O
O
N
H
Br
Br
Br
O
N
20
17
10.3
10.9
15.4
90.7
53.3
42.8
NHMe
NHMe
MeO
Br
N
H
Br
O
N
nt
nt
nt
nt
25.3
41.2
133
75.8
25.6
nt
nt
MeO
Cl
Br
Br
N
Br
OH
Br
N
13
108.2
MeO
Cl
N
nt: not tested.
tested for their antiproliferative activity on human colon cancer
line COLO 205, breast cancer cell line MCF-7, human lung adeno-
carcinoma cell line A549, human acute monocyctic leukemia cell
line THP-1, liver hepatocellular carcinoma cell line HepG2 and
mouse melanoma cell line B16-F10. Both ma’edamines A and B
were found to show significant in vitro cytotoxicity against COLO
205 (IC50 7.9 and 10.3
10.9 M, respectively) and A549 (IC50: 12.2 and 15.4
tively). Ma’edamine A was found to be about equally cytotoxic
on MCF-7 and A-549 cell line compared to etoposide (IC50
16.3 M and 11 M, respectively). Ma’edamines A and B showed
lM, respectively), MCF-7 (IC50: 6.9 and
l
lM, respec-
:
l
l
low activity on THP-1, B16-F10 and HEPG2 cell lines. The interme-
diates 12, 13, 16 and 17 were less potent compared to the
ma’edamines.
Figure 3b. Cells treated with the ma’edamine A (IC50 = 6.93 lMol).The comets with
large tail indicate DNA damage.
The ability of ma’edamines to cause programmed cell death was
assessed by comet assay (Figs.3a–3c),15,21 which is a very sensitive
and most commonly used experiment to find out double strand
breaks in the DNA of eukaryotic cells. The cells treated with the
Figure 3c. Cells treated with the ma’edamine B (IC50 = 10.95 lMol). The comets
with large tail indicate DNA damage.
drug were embedded in low melting agarose immobilized on a
microscopic slide and subjected to cell lysis at alkaline pH followed
by electrophoresis at high pH. The slides were observed under
Figure 3a. The untreated control cells are damage free. The entire DNA is intact
without any double stranded DNA breaks. No tail is observed after comet assay.
Please cite this article in press as: Saha, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.033

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S. Saha et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
assay, trypan blue staining, comet assay and DNA laddering) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.12.033.
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Acknowledgments
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18. The alkylation reaction was done in two conditions: (i) N,N-
dimethylformamide using K2CO3 as base and (ii) 0.5 M NaOH/tetrabutyl-
ammonium bromide/DCM.
We sincerely thank GVK Biosciences Private Limited for finan-
cial support and encouragement. Support from analytical depart-
ment is also acknowledged.
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Supplementary data
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Supplementary data (1H and 13C NMR scans of compound 12,
13, 16, 17, 18, 19, 20 and 23 are available. Data related to MTT
Please cite this article in press as: Saha, S.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2012.12.033