14-membered macrocyclic ring-derived toolbox: The identification of small molecule inhibitors of angiogenesis and early embryo development in zebrafish assay

Article abstract of DOI:10.1021/ol3032126

A highly practical and modular synthesis to obtain a diverse 14-membered ring-based macrocyclic toolbox is achieved. These compounds were further tested in zebrafish assays related to early embryonic development, angiogenesis, and neurogenesis, respective

Full text of DOI:10.1021/ol3032126

ORGANIC
LETTERS
2013
Vol. 15, No. 3
436–439
14-Membered Macrocyclic Ring-Derived
Toolbox: The Identification of Small
Molecule Inhibitors of Angiogenesis and
Early Embryo Development in Zebrafish
Assay
Madhu Aeluri,† Chinmoy Pramanik,†,# Lakshindra Chetia,†,r Naveen Kumar Mallurwar,†
Sridhar Balasubramanian,‡ Gayathri Chandrasekar,§ Satish Srinivas Kitambi,*,§, ,^
and Prabhat Arya*,†
Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli,
Hyderabad 500046, India, Indian Institute of Chemical Technology, Tarnaka,
€
€
€
Hyderabad 500 607, India, School of Life Sciences, Sodertorns Hogskola, Sweden,
and Department of Biosciences and Medical Nutrition and Division of Molecular
Neurobiology, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Sweden
prabhata@drils.org
Received November 22, 2012
ABSTRACT
A highly practical and modular synthesis to obtain a diverse 14-membered ring-based macrocyclic toolbox is achieved. These compounds were
further tested in zebrafish assays related to early embryonic development, angiogenesis, and neurogenesis, respectively. 1.4c was identified as
an antiangiogenesis agent.
The need to assemble a small molecule toolbox with
compounds that are more natural-product-like to search
for modulators of proteinꢀprotein interactions1ꢀ3 and
dissectors of pathways4,5 has grown in recent years. In
particular, interest in the macrocyclic natural products is
rising because they provide diverse functionality and
stereochemical complexity in a conformationally preorga-
nized ring structure.6 These features might be useful for
high affinity and selectivity for protein targets, while
preserving sufficient bioavailability to approach intracel-
lular targets. Despite these valuable characteristics and the
proven success of several marketed macrocycle drugs
derived from natural products, this structural class has
been poorly explored within drug discovery.1 Certainly,
this testifies to the need to develop practical and modular
methods that allow us to build a toolbox with a diverse set
† University of Hyderabad Campus.
‡ Indian Institute of Chemical Technology.
§
€
€
€
Sodertorns Hogskola.
Department of Biosciences and Medical Nutrition, Karolinska Institutet.
^ Division of Molecular Neurobiology, Karolinska Institutet.
# Present address: Emcure Pharmaceuticals, Pune, Maharashtra, India.
r Present address: Syngene International Pvt. Ltd. Bangalore, India.
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(7) Dandapani, S.; Marcaurelle, L. A. Nat. Chem. Biol. 2010, 6, 861.
r
10.1021/ol3032126
Published on Web 01/18/2013
2013 American Chemical Society

be achieved through selective alkylation and amidation,
respectively. For our initial studies, we focused on a
14-membered ring skeleton because there are several examples
of the presence of this ring in a wide variety of bioactive
natural products, such as radicicol,18 pochonin,19 hypo-
themycin,20 aigialomycin, and other related compounds
(structures not shown). Four macrocyclic targets (1.1ꢀ1.4)
were to be obtained from a common starting material
(1.5) which is easy to access, and both enantiomers could
be obtained in several gram quantities in a short duration.
This can further allow us to explore the stereochemical
diversity on a macrocyclic ring.
Scheme 1. A Modular Approach To Access a Diverse Set
of 14-Membered Ring Macrocyclic Compounds
As shown in Scheme 2, 2-nitrobenzaldehyde was con-
verted to an R,β-unsaturated carboxylester by a Hornerꢀ
WadsworthꢀEmmons reaction and then subjected to a
Sharpless asymmetric dihydroxylation reaction, giving an
enantiopure dihydroxyl derivative. Following the aceto-
nide protection of the diol, the carboxylester was then
reduced with lithium borohydride to give primary alcohol
2.2. Primary amine 2.3 was then obtained from 2.2 in four
steps: (i) the conversion of alcohol to azide by mesylation
with methane sulfonyl chloride and then by treating with
sodium azide, (ii) the deprotection of actonide, (iii) 1,2-diol
methylation with methyl iodide, and, finally, (iv) the
reduction of azide by a Staudinger reaction to give primary
amine 2.3. This was converted to a secondary amine by
imino-reduction with isobutyraldehyde, which was then
of macrocyclic compounds to explore their biological
functions.7ꢀ15
With this objective, we were interested in developing a
modular synthesis method to access different types of
14- membered ring macrocyclic compounds as shown in
Scheme 1. In our approach, we were interested in devel-
oping a method that is simple and practical in nature, and
in our design strategy, we can introduce skeletal diversity
and modulate various functional groups, i.e. through D- or
L-amino acids and Sharpless chemistry.16 The proposed
natural product-inspired compounds are more closely
related to natural products in terms of 3D shapes and the
dense display of chiral functional groups. One of the major
advantages is that they are easy to explore in regards to
stereochemical and skeletal diversity and the chemical
space around the scaffold and are easy to synthesize on a
gram scale in a reasonable time scale.7,8,17
In our modular design strategy, we had the option to
bring the amino acid functionality either through the
aromatic amine or from the aliphatic side chain (see
macrocyclic targets 1.1ꢀ1.4). Further, variation in the
side chain, i.e. R2ꢀR4, on the macrocyclic skeleton can also
Scheme 2. Synthesis of 14-Membered Macrocycles 1.1 and 1.2
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Mulrooney, C. A.; Pandya, B. A.; Rowley, A.; Ryba, T. D.; Suh, B. C.;
Wei, J.; Young, D. W.; Akella, L. B.; Ross, N. T.; Zhang, Y. L.; Fass,
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coupled withdifferent N-protectedamino acids (2.6) under
HBTU conditions to obtain compounds 2.4. With com-
pounds having NCbz as the protecting group, the nitro
reduction and NCbz removal were performed in a single
(15) Peng, L. F.; Stanton, B. Z.; Maloof, N.; Wang, X.; Schreiber,
S. L. Bioorg. Med. Chem. Lett. 2009, 19, 6319.
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Org. Lett., Vol. 15, No. 3, 2013
437

step with 10% Pd/C under hydrogen conditions to obtain
diamines, which were then converted to bisamides on
reaction with different acid chlorides. Compounds having
NFmoc were converted into 2.5 in four steps: (i) the
removal of NFmoc with DBU, (ii) amidation with differ-
ent acid chlorides, (iii) reduction of nitro to amine with
10% Pd/C, and (iv) amidation with different acid chlor-
ides. Macrocyclic compounds, 1.1, were obtained from the
corresponding bisamides in two steps, i.e. bisallylation
with allylbromide and NaH followed by ring-closing
metathesis21ꢀ23 using the Grubbs second generation cata-
lyst (G-II). In this series, eight macrocyclic compounds
were synthesized, and in all cases, the trans olefin geometry
was obtained as determined by NMR (see the Supporting
Information (SI)).
Scheme 3. Synthesis of 14-Membered Macrocycles 1.3 and 1.4
In a similar manner, using 2-benzyloxybenzaldehyde as
the starting material, we completed the synthesis of eight
macrocyclic compounds 1.2. As before, the trans olefin
geometry was obtained following the “stitching technol-
ogy”. The detailed synthesis for 1.2 is provided in the SI. In
one case (1.2a), we could further obtain the X-ray to
confirm the macrocyclic skeleton.
In another series, macrocycles 1.3 (Scheme 3) were
obtained from the key intermediate 2.3 in eight steps as
follows. Primary amine 2.3 was converted to an amide with
benzoyl chloride and then by allylation on amide ꢀNH
followed by the reduction of an aromatic nitro to give the
aromatic amine 3.2. This was coupled with N-protected
amino acids (3.6) using EDC•HCl and then subjected to
NFmoc removal to obtain 3.3. Monoallylation (allyl bro-
mide, MeOH) of 3.3 was followed by an amidation to
obtain the key bisallyl product 3.4, needed for the stitching
technology. Four macrocyclic compounds 1.3 were ob-
tained using the ring-closing technology (Grubbs second
generation catalyst, G-II). In this series, although we ob-
tained a single isomer (determined by HPLC-MS), the olefin
geometry remains to be determined. Similarly, four macro-
cycle compounds 1.4 were obtained (see the SI), and in one
case, we could obtain the X-ray of the reduced double bond
macrocyclic product 3.5 to further confirm our assignments.
A small molecule toolbox containing 85 compounds was
then subjected to a search for chemical probes affecting
epiboly during early embryonic development,24ꢀ26 angio-
genesis,27,28 and neurogenesis29 in zebrafish embryo-based
assays.30,31 These assays are well-documented in the
literature,28,32ꢀ34 and the detailed experimental procedure
is provided in the SI. It was interesting to observe that
among all the compounds tested from this collection, we
identifieda novel small molecule (1.4c, Figure 1) thatspeci-
fically inhibited the trunk angiogenesis (see Figure 1B and
C) at 2.5 μM without affecting either epiboly or neurogen-
esis. Embryos exposed to 2.5 μM of this small molecule
(1.4c) displayed normal epiboly movement and neurogen-
esis, and an overall embryonic development was also not
affected. The embryos showed a complete lack of migra-
tion of early endothelial cells (i.e., tip and stalk cells) to
form an early intersegmental vessel of the trunk.
In another parallel study, we identified four active
compounds (S27d, 2.5c and 1.1c, 1.2f) that inhibited epi-
boly cell movements during early embryonic development
(Figure2). Thedelayinepiboly wasclearly seeninembryos
exposed to 5.0 μM of the small molecules; however these
embryos did complete epiboly and developed normally
without any visible effects on angiogenesis and neurogen-
esis. Athigherconcentrations, suchas10.0μM, epibolydid
not begin leading to the lethality of the embryo. The
mechanism underlying the inhibition of angiogenesis and
epiboly will be characterized using both zebrafish and
other cell based assays.
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To summarize, we report a practical and modular
approach for accessing a diverse set of 14-membered ring
(27) Konantz, M.; Balci, T. B.; Hartwig, U. F.; Dellaire, G.; Andre,
M. C.; Berman, J. N.; Lengerke, C. Ann. N.Y. Acad. Sci. 2012, 1266, 124.
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(29) Kitambi, S. S.; Malicki, J. J. Developmental dynamics: an official
publication of the American Association of Anatomists 2008, 237, 3870.
(30) Kitambi, S. S.; Nilsson, E. S.; Sekyrova, P.; Ibarra, C.; Tekeoh,
G. N.; Andang, M.; Ernfors, P.; Uhlen, P. BMC Physiology 2012, 12, 3.
(31) Murphey, R. D.; Zon, L. I. Methods 2006, 39, 255.
(32) Zhang, Q.; Li, Q.; Chen, Y.; Huang, X.; Yang, I. H.; Cao, L.;
Wu, W. K.; Tan, H. M. Frontiers in Bioscience 2012, 4, 2525.
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Gentry, P. R.; Hopkins, C. R.; Lindsley, C. W.; Hong, C. C. ACS Chem.
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Org. Lett., Vol. 15, No. 3, 2013

explore its value. The presence of two contiguous stereo-
genichydroxyl group derivativesandanaminoacidmoiety
(note: in the present study only natural amino acids were
utilized) in the macrocyclic ring architecture allow access
to this unique set of compounds. With the goal of going
beyond the conventional chemical space in the drug dis-
covery arena where most compounds are rich in sp2
character, the present method provides a good entry to
access a diverse set of 14-membered macrocyclic com-
pounds with variation in the display of different functional
groups. Furthermore, toexplore their biological effects, we
utilized zebrafish embryonic screening assays. These rapid
assay procedures allowed us to quickly identify the effects
of small molecules on various biological processes. The
effects produced by these novel molecules provided a
platform for using chemical biology approaches to under-
stand basic biological processes such as epiboly and angio-
genesis. Extensive validation procedures need to be
developed to characterize the effect produced and to
elucidate the mechanism of action. Further, work is
needed to understand the deep impact of these functional
small molecules in the context of developing a new class of
inhibitors of angiogenesis as well as the inhibitors of early
embryonic development.
Figure 1. Zebrafish angiogenesis assay: (A) Wild-type zebra fish
embryo at 30 hpf of development, region zoomed in panels B
and C is shown by a yellow box; (B) zoom section of wild-type or
vehicle treated embryo; (C) zoom section after treatment with
compound 1.4c.
Acknowledgment. This work was supported by DST
and DBT grants to P.A. M.A. and N.K.M. thank CSIR
and UGC funding agencies for the award of PhD fellow-
ships. An earlier portion of this work was carried out by
C.P. and L.C. during their postdoctoral tenure with P.A. at
the National Research Council of Canada (NRCC) and
Ontario Institute of Cancer Research (OICR). Don Leek
and Malgosia Darozewska (both at NRCC) are thanked
for the analytical services provided. We also thank the ILS
analytical team for providing excellent HPLC-MS and
€
NMR support. S.K. thanks Sodertorns Hogskola for the
financial support to perform zebrafish assays.
€
€
Figure 2. Zebrafish early embryonic development assay: (A)
DMSO exposed embryos at 10 hpf of development; (B) small
molecule exposed embryos causing a delay in epiboly.
Supporting Information Available. The detailed synthe-
sis procedure and characterization of all new com-
pounds are thoroughly provided. This material is avail-
able free of charge via the Internet at http://pubs.
acs.org.
macrocyclic compounds. The ease of the synthesis and the
reported modular methodology are the two attractive
features in assembling a macrocyclic chemical toolbox to
The authors declare no competing financial interest.
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