Synthesis and structure-activity relationship of botryllamides that block the ABCG2 multidrug transporter

Article abstract of DOI:10.1016/j.bmcl.2010.01.016

In previous work, botryllamides discovered from the marine ascidian Botryllus tyreus were characterized as selective inhibitors of the ABCG2 multidrug transporter. However, the structural basis for this activity could not be established. In this study, bo

Full text of DOI:10.1016/j.bmcl.2010.01.016

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1330–1333
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship of botryllamides that block
the ABCG2 multidrug transporter
a
b
b,c
Kentaro Takada a, , Nobutaka Imamura , Kirk R. Gustafson , Curtis J. Henrich
*
a Laboratory of Microbial Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan
b Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Building 1052, Room 201, Frederick, MD 21702-1201, USA
c Basic Research Program, SAIC-Frederick, Inc., Building 538, Room 141D, Frederick, MD 21702-1201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In previous work, botryllamides discovered from the marine ascidian Botryllus tyreus were characterized
as selective inhibitors of the ABCG2 multidrug transporter. However, the structural basis for this activity
could not be established. In this study, botryllamide F, the core botryllamide structure, and botryllamide
G, the most potent botryllamide ABCG2 inhibitor, were synthesized along with a series of structural
variants for evaluation of structure–activity relationships. The biological activity of synthetic botrylla-
mide analogs implied that the 2-methoxy-p-coumaric acid portion, and the degree of double bond
conjugation within this group, were critical for inhibition of ABCG2. However, variations in the substit-
uents on the two aryl groups did not appear to significantly impact the potency or degree of inhibition.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 9 December 2009
Revised 29 December 2009
Accepted 5 January 2010
Available online 11 January 2010
Keywords:
Natural products
ABCG2 blocker
Structure–activity relationships
Synthesis
ABCG2 is a member of the ATP-binding cassette (ABC) family of
transporters, which pump a wide range of chemically diverse sub-
strates out of cells using energy from ATP hydrolysis.1–3 The over-
expression of ABCG2 in cancer cells may play an important role in
exporting chemotherapeutic agents, such as mitoxantrone, topo-
tecan, irinotecan, doxorubicin, SN-38, and flavopiridol, resulting
in cellular resistance to these agents. Accordingly, compounds that
inhibit ABCG2 function may prove to be useful therapeutic agents.
Although a number of ABCG2 inhibitors have been described, they
lack potency or specificity or have toxic effects, and none have
been developed as clinical agents.
P-gp. The difference in selectivity appears to be based on a very
small structural difference (the presence or absence of an O-methyl
group at C15). However, the structural basis for the range of poten-
cies against ABCG2 could not be established based on the naturally
occurring botryllamides that were obtained.
R
15
HO
O
10
2
R
N
H
7
OMe
OH
We recently developed a high-throughput cell-based assay4 to
screen compounds for inhibition of ABCG2 activity by blocking
1: R=H Botryllamide F
2: R=Br Botryllamide G
efflux of the fluorescent substrate pheophorbide-a (PhA).5,6
A
new structural class of ABCG2 inhibitors was characterized, botry-
llamides A–J from the Micronesian colonial ascidian Botryllus tyre-
us.7 Two of these, I and J, were also novel structures. Despite only
modest structural differences, the botryllamides showed variable
activity against ABCG2 and variable specificity toward ABCG2 rel-
ative to other multidrug transporters [P-glycoprotein (P-gp) and
MRP1]. For example, botryllamide G inhibited the efflux of PhA
O
OH
R1
R2
HO
OH
3:R1=R2=OMe
Curcumin
4:R1=H, R2=OMe Demethoxy curcumin
out of ABCG2-overexpressing cells with an IC50 value of 6.9
but not the efflux of rhodamine out of P-gp-overexpressing cells
at 50 M. In contrast, botryllamide A was much less potent against
ABCG2 (IC50 = 33 M), but inhibited the export of rhodamine by
lM,
5:R1=R2=H
Bis-demethoxy curcumin
l
To explore structure–activity relationships (SAR) among the botry-
llamides with regard to ABCG2, additional structural analogs were
synthesized. Given the relatively low yields that were obtained
from natural product extracts, a synthetic approach to the botrylla-
mides would also be useful for generation of sufficient material to
l
* Corresponding author. Tel.: +81 77 561 2598; fax: +81 77 561 5203.
E-mail address: takada@ph.ritsumei.ac.jp (K. Takada).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.016

K. Takada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1330–1333
1331
support further biological characterization and in vivo testing of
left aryl group
HO
these compounds. Botryllamide F (1), the core botryllamide struc-
ture, was selected as the initial synthetic target. We utilized a strat-
egy in which octopamine was condensed with the C1–C9 portion of
1 to generate an amide bond, and then dehydration of the adjacent
hydroxyl group provided the key enamine amide functionality
(Scheme 1). The C1–C9 fragment, 2-methoxy-p-coumaric acid (8),8
was synthesized from methylmethoxyacetate (7) and 4-hydroxy-
benzaldehyde (6). Then 8 was coupled with octopamine hydrochlo-
ride, and the three hydroxyl groups were acetylated. The hydroxyl
group at C11 was dehydrated with a catalytic amount of K2CO3 to
give 1.9,10 The 1H and 13C NMR spectrum of 1 showed good agree-
ment with those of natural botryllamide F. Subsequently, we syn-
O
right aryl group
10
4
12
1
N
H
OMe
OH
linker
Figure 1. Strategy for the SAR study of botryllamides.
ecules and their inhibitory activity toward ABCG2. We synthesized
three analogs: 2,3-dihydro botryllamide F (15), 10,11-dihydro bot-
ryllamide F (16), and 2,3,10,11-tetrahydro botryllamide F (17). To
synthesize 15, tyramine hydrochloride was coupled with 8 instead
of octopamine hydrochloride, and 16 and 17 were prepared by the
hydrogenation of 8 and 1, respectively.
thesized botryllamide
G (2), which is the most potent and
selective inhibitor of the ABCG2 transporter. The reactions for the
synthesis of 2 were similar to those described for 1, apart from
using bis-brominated octopamine.11 The 1H and 13C NMR spectrum
of 2 also showed good agreement with those of natural botrylla-
mide G. Both synthetic botryllamides had comparable potency in
the PhA assay as compared to the natural products (data not
shown).
In selecting synthetic botryllamide analogs for an SAR study, we
took into account the structure of the curcumins (3–5), which have
also been reported to inhibit the transport of mitoxantrone and
PhA out of ABCG2-overexpressing cells.12 The curcumins and the
botryllamides have a common biochemical profile of stimulating
the ATPase activity of ABCG2. This effect is in contrast to many
ABCG2 inhibitors, which reduce ATP hydrolysis by ABCG2.7,12 Cur-
cumins are the major curcuminoids found in turmeric powder and
their structures consist of two hydroxyl substituted phenyl rings
joined by an acyclic linker. The general structural features of the
botryllamides are quite similar to the curcumins, since they also
have two aryl rings joined by an acyclic linker. Differences between
these two families of compounds are seen in both the length and
functional group composition of the linkers. Botryllamides have
ABCG2 inhibitory activity was evaluated by the cellular accu-
mulation of the ABCG2 specific substrate PhA in cells that over ex-
pressed the transporter, as described previously.4,13 Potency (IC50
)
for each compound was estimated from dose–response curves and
shown in Table 1. Although a high degree of conjugation in the lin-
ker is a common structural feature of both the botryllamides and
curcumins, the activities of the three analogs indicated that only
maintenance of the D2,3 double bond was essential to inhibit
ABCG2. The desmethoxy analog (18) was inactive at 100 lM, sug-
gesting that the methoxy group at C2 was also necessary for activ-
ity. Synthetic intermediates 8 and 9 were inactive, suggesting that
the most significant moiety for inhibitory activity was the 2-meth-
oxy-p-coumaric acid (C1–C9) portion; moreover, the C10–C17 half-
styrene moiety in the botryllamides may play a role as an anchor.
Next, we evaluated the effect of different functional group sub-
stitutions on the C4–C9 benzene ring, because the activity data
suggested the 2-methoxy-p-coumaric acid portion of the botrylla-
mides was important in interacting with the ABCG2 protein. While
the natural botryllamides we isolated possessed a variety of sub-
stituents on the C12–C17 (left) aryl ring, the substitution pattern
of the C4–C9 (right) aryl ring was generally conserved. Variations
in the substituents on the C12–C17 ring did not correlate with
ABCG2 inhibition, but they may contribute to specificity. Next,
we modified the functional groups on the C4–C9 ring and tested
their inhibitory activity against ABCG2 (Table 2).
an enamine amide group conjugated to the enol ether of an a-keto
group, while the cucurmins have a cross conjugated enolized b-
diketone functionality. In addition, the cucurmins have a seven
carbon long linker that is pseudosymmetrical due to enol tauto-
merization, while the botryllamides have a nonsymmetrical and
shorter linker comprised of five carbons and one nitrogen. Differ-
ences in aryl group substitution patterns are also found between
the botryllamides and the curcumins.
We used various starting materials instead of 4-hydroxybenzal-
dehyde to synthesize the analogs 19–24. The deshydroxyl deriva-
tive 19 retained inhibitory activity, indicating that the phenolic
For the SAR study (Fig. 1), we initially examined the relationship
between the degree of conjugation in the linker portion of the mol-
O
O
OMe
MeO
+
H
a
HO
b
O
OMe
OH
OH
6
7
8
R1
R1
HO
R1
HO
R1
O
O
d
N
H
N
H
R2
OMe
OMe
OH
OH
9: R1=H, R2=OH
10: R1=Br, R2=OH
13: R1=H
14: R1=Br
c
11: R1=H, R2=OAc
12: R1=Br, R2=OAc
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, reflux, overnight, 75%; (b) octopamine HCl (bis-brominated octopamine for 10), WSCI, HOBt, Et3N, rt, 4 h; (c) Ac2O,
Pyridine, rt, 2 h, two steps 58%; (d) K2CO3, DMSO, 98 °C, 2 h 45%.

1332
K. Takada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1330–1333
Table 2 (continued)
Structure
Table 1
IC50
(lM)
SDa
ABCG2 inhibitory activity of analogs 8, 9 and 15–18
HO
Structure
IC50
(lM)
SDa
O
HO
N
H
O
>100
OMe
OBn
N
>100
25.2
H
24
Standard deviation; n = 3–4.
OMe
OH
15
a
HO
O
N
1.7
hydroxyl group on C7 was not a required functional group for
activity. The observed inhibitory activity of the 6,7-dihydroxyl
derivative 21 and the 6,8-dibromo derivative 22 suggested that
the binding region on ABCG2 that the C4–C10 ring interacts with
is large enough to accommodate additional functionalities. How-
ever, the O-benzyl group of compound 24 was too large for ade-
quate binding to the protein, judging from its lack of inhibitory
activity. Based on variations of the substituents on the C12–C17
aryl ring observed with the natural botryllamides, and their broad
ABCG2 inhibitory activities, an ABCG2 binding site for this portion
of the molecule would appear to be relatively large and structurally
permissive.
In this study, we established a simple and readily scalable syn-
thetic route to botryllamide F (1) and G (2), and prepared a series of
botryllamide analogs. The ABCG2 inhibitory activities of the botry-
llamide analogs indicated that conjugation through C1 to C9 was
necessary for the activity; however, extended conjugation through
C10 to C17 was not required. Furthermore, the SAR study sug-
gested that two distinct binding interactions occur with the ABCG2
protein. Thus, this synthetic study provides a means for the contin-
ued preclinical evaluation of the botryllamides and it may lead to
the development of more potent and selective ABCG2 inhibitors
as potential therapeutic agents.
H
OMe
OH
16
HO
O
N
>100
>100
>100
H
OMe
OH
17
HO
O
N
H
OH
18
HO
O
N
H
OH
OMe
OH
9
O
HO
>100
OMe
OH
8
a
Standard deviation; n = 3–4.
Acknowledgements
We are grateful to Dr. Hiroshi Hirota, RIKEN, JAPAN, for helpful
suggestions. This project has been partly supported by Compre-
hensive Support Programs for Creation of Regional Innovation from
Japan Science and Technology Agency. This project has also been
funded in whole or in part with federal funds from the National
Cancer Institute, National Institutes of Health, under contract
HHSN261200800001E. The content of this publication does not
necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by the
U.S. Government. This research was supported in part by the Intra-
mural Research Program of the NIH, National Cancer Institute, Cen-
ter for Cancer Research.
Table 2
ABCG2 inhibitory activity of analogs 19–24
Structure
IC50
(lM)
SDa
1.0
HO
O
N
15.4
H
OMe
19
HO
O
N
6.4
0.8
1.2
H
OMe
OMe
20
HO
HO
O
Supplementary data
OH
OH
N
H
18.8
OMe
OMe
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.016.
21
O
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Br
N
H
11.1
23.2
1.2
2.5
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Then 8 was coupled with octopamine hydrochloride, and the three hydroxyl
groups were acetylated. After the purification of the triacetate 11 on SiO2, the
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