Synthesis and SAR studies of marine natural products ma'edamines A, B and their analogues

Article abstract of DOI:10.1016/j.bmcl.2013.07.017

The synthesis of several analogues of ma'edamines A and B are reported. The synthesized compounds were tested on hormone receptor positive and HER2 positive breast cancer cell lines, by MTT assay. MED-114, 115, 117, 119, 120, 124, 128 and 131 were found to be equally active as Lapatinib on HER2 +ve cell line SKBR3.

Full text of DOI:10.1016/j.bmcl.2013.07.017

Accepted Manuscript
Synthesis and SAR studies of marine natural products ma’edamines A, B and
their analogues
Sanjay Saha, Ch. Venkata Ramana Reddy, Shili Xu, Saranya Sankar, Nouri
Neamati, Balaram Patro
PII:
S0960-894X(13)00862-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.07.017
BMCL 20682
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
19 June 2013
10 July 2013
12 July 2013
Please cite this article as: Saha, S., Venkata Ramana Reddy, Ch., Xu, S., Sankar, S., Neamati, N., Patro, B., Synthesis
and SAR studies of marine natural products ma’edamines A, B and their analogues, Bioorganic & Medicinal
Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.07.017
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Synthesis and SAR studies of marine natural
products ma’edamines A, B and their
analogues
Leave this area blank for abstract info.
Sanjay Sahaa,b, Ch. Venkata Ramana Reddyb,
Shili Xuc, Saranya Sankarc, Nouri Neamatic, Balaram Patroa*
aGVK Biosciences Pvt. Ltd. Medicinal Chemistry Division, Nacharam, Hyderabad, India; b Department of
Chemistry, JNT University, Kukatpally, Hyderabad, India; cDepartment of Pharmacology and Pharmaceutical
Sciences, School of Pharmacy, University of Southern California.
O
Br
H
N
n
R
23 compounds
O
R
R1
R2
O
R1
+
BocHN
N
OH
NH3 Br-
+
O
NR3R4
O
n
R2
The synthesis of several analogues of ma’edamines A and B are reported. The synthesized compounds were tested on hormone
receptor positive and HER2 positive breast cancer cell lines by MTT assay. MED-114, 115, 117, 119, 120, 124, 128 and 131 were
found to be equally active as Lapatinib on HER2 +ve cell line SKBR3.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and SAR studies of marine natural products ma’edamines A, B and
their analogues.
Sanjay Sahaa,b, Ch. Venkata Ramana Reddyb, Shili Xuc, Saranya Sankarc, Nouri Neamatic,
Balaram Patroa*
aGVK Biosciences Private Limited, Medicinal Chemistry Division, 28A, IDA, Nacharam, Hyderabad,500076, India
bDepartment of Chemistry, Jawharlal Nehru Technological University, Kukatpally, Hyderabad, 500085, India
cDepartment of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The synthesis of several analogues of ma’edamines A and B are reported. The
synthesized compounds were tested on hormone receptor positive and HER2 positive
breast cancer cell lines, by MTT assay. MED-114, 115, 117, 119, 120, 124, 128 and
131 were found to be equally active as Lapatinib on HER2 +ve cell line SKBR3.
Available online
Keywords:
Keyword_1
Keyword_2
Keyword_3
Keyword_4
Keyword_5
———
*Corresponding author. Tel.: +091-40-66281666; fax: +091-40-66281505; e-mail: balaram.patro@gvkbio.com; b_patro30@hotmail.com;

The marine world is a source of excellent biodiversity which
translates into great chemical diversity as well. Eight compounds
derived from marine source have been approved till date by the
FDA or EMEA for various disorders. They are Cephalosporin C,
Cytarabine (Ara-C), Vidrabine (Ara-A), Ziconotide (Prialt),
omega-3-acid ethyl esters (Lovaza), ET-743 (Yondelis), E7389
(Halaven) and Brentuximab vedotin (SGN-35).1 A recent review
gives an account of on-going efforts in marine pharmacology and
anti-cancer drug discovery from marine natural products which
are in various phases of clinical development.2 This includes:
bryostatin 1, dolastatin 10, aplidine, kahalalide F, squalamine,
discodermolide, hemiasterlin analogue, bengamide analogue,
cemadotin and eulotherabin. Excellent reviews featuring the
discovery of antiviral and anti-malarial compounds from marine
source have also appeared in literature.3, 4
Figure 1: Design considerations and parameters: a:
Replacement of the 3-bromo-4-methoxyphenol group; b:
replacement of the 3,5-dibromophenyl group; c: variation of
the number of c-atoms connecting the oxygen and the N
atoms; d: replacing the substitution on nitrogen or e: changing
the bromo or the methoxy substituents.
O
Br
n
R
R1
R2
O
BocHN
OH
A
K
O
NH3+Br-
Figure 2: Key Building blocks required for synthesis
Ma’edamines A (Med-111) and B (Med-114) (Table 1) which
have a unique pyrazine-2-(1H)-one core structure was isolated by
Kobayashi and co-workers from a marine sponge Suberea sp.5
Kobayashi et. al. have reported cytotoxic activity of Med-111 and
Med-114 in human and murine tumor cell lines and c-erbB-2
kinase inhibition (IC50: 3.9-6.7 µg/mL for ma’edamine A) in
enzymatic assays.5 We have recently reported the total synthesis
of ma’edamines A and B by two different approaches.6
Ma’edamines A and B were found to be cytotoxic on 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). We report herein the
synthesis of several analogues of ma’edamines and their
biological evaluation for antiproliferative activity on breast
cancer cell lines SKBR3 and T47D. Compounds Med-114, 115,
117, 119, 120, 124, 128 and 131 were found to have
cyctotoxicity with IC50 values in the range of 3.5-8.15 µM which
is comparable to HER2 inhibitor lapatinib.7
The synthesis of ma’edamines A and B was achieved via two
different approaches.6 The first approach involved coupling an α-
keto acid with an α-amino ketone in a key step, followed by
cyclization of the product with ammonium acetate which resulted
in formation of the 2-[1H]-pyrazinone core of ma’edamines. In
the second approach an α-amino acid was coupled with an α-
amino ketone and the resulting product was de-protected,
cyclized and oxidized to give the ma’eadamine core (Scheme 2).
The potent in-vitro cyctotoxicity of ma’edamines on breast
cancer, lung cancer and colon cancer cell lines prompted us to
synthesise several analogues. In the present study, the second
approach was followed to synthesize analogues of ma’edamines.
The design of analogues was based on intuitive modification of
the ma’edamines template. The structure of ma’edamine A was
divided into two hydrophobic regions A and B around the
pyrazin-2[1H]-one ring as shown in Figure 1. The following
modifications were done: substituents on the ring in A region
(designated as E) was modified; the ring in A region was
replaced with different aliphatic and aromatic residues; the
substituents on the ring in region B was modified; the number of
carbon atoms (designated as n) connecting the oxygen and
nitrogen (designated as D) was varied between 2 and 4; and the
substituent on nitrogen (region D) was modified.
Following amino acid building blocks were used for synthesis
of ma’edamines analogues (Figure 3). The compounds a2 and
a3 were prepared by bromination and chlorination
respectively, of L-4-methoxyphenyl alanine, by reported
methods followed
by N-protection
with di-tert-
butyldicarbonate.8, 9All other amino acid derivatives were
obtained from commercial sources and used as such.
Br
Cl
H
H
H
N
N
N
(S)
Boc
(S)
(S)
Boc
Boc
MeO
MeO
O
OH
O
O
OH
OH
A3
A1
A2
H
H
N
N
(S)
H
N
Boc
MeO
(S)
Boc
O
(S)
O
Boc
OH
HN
OH
A5
O
A4
A7
A6
OH
H
N
(S)
Boc
HO
O
OH
Figure 3: α-amino acid building blocks
A key reaction step in the synthesis of the α-Amino ketones
building blocks (Figure 4) was a Delepine reaction of an α-
10
bromoketone compound 4 (Scheme 1).
The synthesis was
started with bromination of 4-hydroxyacetophenone with
pyridinium bromochromate which afforded 3,5-dibromo-4-
hydroxyacetophenone in 93 % yield.11 Chlorination of 4-
hydroxyacetophenone was done by bubbling chlorine gas
through its solution in acetic acid and water to afford 3,5-
dichloro-4-hydroxyacetophenone in 30 % yield.12 Alkylation of
the resulting di-halo products 2 with 1,2-diboroethane / 1,3-
dibromopropane / 1,4-dibromobutane in DMF solvent using
potassium carbonate as a base afforded the O-alkylated product
3. The acetyl group of the resulting product 3 was brominated
using bromine in chloroform at room temperature. A Delépine
reaction on the intermediate 4 which involved reaction with
hexamethylenetetramine followed by hydrolysis of the resulting
salt with HBr, afforded the desired α-amino ketone K.
E
A
n
B
Br
Br
MeO
O
Br
N
NMe2
D
O
N
H

O
O
Br
n
R
O
R1
R2
R
N
H
a
b
O
NH
+
Boc
BocHN
OH
R1
R2
NH3+Br-
A
6
O
O
C
c
K
O
n
Br
H
N
ClH.H2N
O
NH
R
O
+
R1
O
R
N
7
D
8
Br
n
R2
R2
R1
Scheme 1: Reagents and Conditions: a) pyridinium
bromochromate, AcOH, 40 °C, 2h, ~70%; or Cl2, AcOH, H2O,
~75%; b) BrCH2(CH2)nCH2Br, (n=0-2), K2CO3, DMF, rt, 36h,
60%; c) Br2, CHCl3, rt, 2h, 95%; d) Hexamethylenetetramine,
CHCl3, rt, 18h; e) MeOH, HBr (aq.), 48h, 66% for two steps.
O
H
N
O
R
n
R1
O
Br
N
E
NR3R4
9
n
R2
Scheme 2 : Reagents and Conditions: a) HATU, TEA, DMF, 4h,
40-60%; b) 4M HCl in 1,4 - dioxane, rt, 4h; c) Amine in THF, rt,
16- 24h, ~30-40 % for two steps.
The analogues of ma’edamines A and B were synthesized by a
route shown in Scheme 2. The first step in this route is based on
the coupling of an α-amino acid A with an α-amino ketone
derivative K mediated by HATU which afforded us the amide 6.
De-protection of the amide with 4M HCl in 1,4-dioxane at room
temperature afforded a mixture of de-protected derivative 7 and
cyclized compound 8. Treatment of this mixture with the
appropriate amine offered us the aromatized compound 9 suitably
substituted by an alkyl amino group. All the synthesized
compounds were purified by flash column chromatography over
silica gel followed by crystallization. The prepared analogues
Figure 4: Synthesized α-Amino ketone building blocks.
1
were characterized by H NMR and HPLC-MS and obtained in
sufficiently pure form (>95 %). They were screened for anti-
proliferative activity by MTT (3,4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay13 on SKBR3 [estrogen
receptor (ER) -ve, progesterone receptor (PR) –ve and human
epidermal growth factor receptor (HER2) +ve ], T47D (ER +ve,
PR +ve and HER2 -ve) and CAMA 1 (ER +ve, PR –ve and HER
2 -ve) cell lines. 14 The IC50 values are reported in Table 1.

Table 1: Synthesized compounds and their anti-proliferative activity against breast cancer cell lines
IC50 (µM)
Compound
MED-
Structure
SKBR3
T47D
CAMA1
110
>10
>10
>10
>10
111
114
115
1166
117
118
119
120
121
123
124
125
127
128
>10
6.17±0.27
4±0.80
>10
>10
5.77±0.37
6±0.0
>10
6.18±1.45
6±0.0
>10
5.5±0.0
> 10
6.33±0.53
>10
7.10±0.96
>10
5.73±1.24
8.10±0.21
> 10
5.67±1.87
6.18±1.45
>10
>10
>10
>10
7±0.0
6.33±1.07
4±2.4
>10
4.33±0.53
> 10
5.77±0.37
>10
>10
> 10
>10
>10
6±0.0
>10
5.23±0.05

129
> 10
> 10
>10
>10
>10
130
>10
131
5.67±0.53
> 10
4.77±0.03
6.67±1.07
>10
132
>10
>10
>10
-
133
> 10
>10
134
> 10
>10
137
>20
>20
138
>20
-
>20
Lapatinib
5.05±0.28
>10
>10
The cytotoxicity of compounds MED-114 (ma’edamine B), 115,
117, 119, 120, 124, 128 and 131 on SKBR3 cell lines were
comparable to Lapatinib (IC50 5.05±0.28 µM), a compound
approved by FDA for HER2 targeted therapy of breast cancer.
MED-115 and MED-124 were found to be the most potent
compounds in this series with a mean IC50 4 µM and 4.33 µM
respectively against SKBR3 cell line. MED-117, a compound
without a bromo and a methoxy group on the phenyl ring (in A
region) was also quite potent (IC50 5.5 µM, SKBR3) suggesting
that these groups on the phenyl ring weren’t absolutely essential
for activity.
When the hydrophobic phenyl group of MED-111 (ma’edamine
A) was replaced by an indole in compound MED-123 (IC50 ~7
µM, SKBR3) and MED-124 (IC504.33±0.53 µM, SKBR3) the
activity was retained. However when the aromatic group was
replaced with an aliphatic group in MED-125 the resulting
compound was inactive. Replacement of the bromo groups of
MED-111 with chloro groups (MED-121) didn’t result in any
improvement in activity. De-methylation of MED-111 resulted in
restoration of antiproliferative activity in compound MED-131
(IC50 5.67±0.53 µM, SKBR3). The replacement of methyl groups
with ethyl (MED-128) also resulted in restoration of activity
(IC506µM, SKBR3).
Completely de-brominated compounds MED-129, MED-130
(IC50>10 µM) and MED-134 (IC50>10 µM) didn’t show any
improvement in activity over their brominated counterparts MED
111 (IC50> 10 µM) and MED 133 (IC50>10 µM). De-bromination
resulted in loss of activity in MED-137 (IC50>20 µM) and MED-
138 (IC50>20 µM) compared to their halogenated couterparts
MED-123 (IC50 ~7 µM, SKBR3) and MED-124 (IC504.33±0.53
µM, SKBR3).
The amino-alkyl side chain is required for activity since removal
of side chain of MED-115 (IC50 4±0.80 µM, SKBR3) resulted in
complete loss of activity in MED-116 (IC50 >10 µM, SKBR3).
Reducing the length of the amino-alkyl side chain of MED-111
to two carbons resulted in improvement of activity in MED 119
(IC505.73±1.24 µM, SKBR3), whereas increasing the chain
length to four carbons did not improve the activity in MED-133
(IC50 >10 µM, SKBR3). Reduction of chain length of active
compound Med-114 (IC50 6.17±0.27 µM, SKBR3) did not
changed its activity significantly in MED-120 (IC50 8.10±0.21
µM, SKBR3).
MED-114, 115, 117, 119, 120, 123, 124 and 131 were also had
good cyctotoxicity on hormone receptor positive cell line T47D
with IC50 values in the range of 4-6.33 µM (Table 1).

In conclusion, we have prepared 23 analogues of ma’edamines
A and B and tested them on three breast cancer cell lines
representing hormone receptor positive and HER2 positive breast
cancer. It is evident from the cytotoxicity data that MED-114,
115, 117, 119, 120, 123, 124, 128 and 131 were equally active as
Lapatinib on HER2 +ve cell line SKBR3. MED-114, 115, 117,
124 and 131 were active on all three types of breast cancer cell
lines.
Acknowledgments
We sincerely thank GVK Biosciences Private Limited for
financial support and encouragement. Support from analytical
department is also acknowledged.
References and notes
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Supplementary Material
1H NMR scans and mass spectral scans of all newly synthesized
compounds are available.