Macrocyclic glycohybrid toolbox identifies novel antiangiogenesis agents from zebrafish assay

Article abstract of DOI:10.1021/ol3032297

A practical and modular approach to obtain a diverse set of 14-membered macrocyclic compounds from carbohydrates is developed that utilizes functional groups at C-1 and C-5. The evaluation of this toolbox in various zebrafish assays led to the identificat

Full text of DOI:10.1021/ol3032297

ORGANIC
LETTERS
2013
Vol. 15, No. 3
432–435
Macrocyclic Glycohybrid Toolbox
Identifies Novel Antiangiogenesis Agents
from Zebrafish Assay
Bhanudas Dasari,† Srinivas Jogula,† Ramdas Borhade,# Sridhar Balasubramanian,‡
Gayathri Chandrasekar,§ Satish Srinivas Kitambi,*,§, ,^ and Prabhat Arya*,†
Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli,
Hyderabad 500 046, 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, Division of Molecular Neurobiology,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Sweden
prabhata@drils.org
Received November 23, 2012
ABSTRACT
A practical and modular approach to obtain a diverse set of 14-membered macrocyclic compounds from carbohydrates is developed that utilizes
functional groups at C-1 and C-5. The evaluation of this toolbox in various zebrafish assays led to the identification of 2.7f as an antiangiogenesis
agent.
The growing interest in macromolecular (i.e., proteinÀ
protein,1,2 DNA/RNAÀprotein) interactions,3 and in sig-
naling pathways4 is challenging our current thinking in the
drug discovery arena.5 This is also creating new opportu-
nities to develop novel chemical approaches to allow us to
build unique chemical tool sets that are more closely
related to bioactive natural products or derived from their
inspiration.6,7 This is largely due to the fact that natural
† 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: Sai Advantium Pharma Ltd., Pune, Maharashtra, India.
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r
10.1021/ol3032297
Published on Web 01/18/2013
2013 American Chemical Society

products in general display 3D shapes and possess several
chiral functional groups that are a good source of small
molecule modulators of proteinÀprotein, DNA/RNAÀ
protein interactions.1,2,8 In addition, interest is also rising
in emerging screening approaches, such as the use of
cellular phenotypes5a and in vivo models (i.e., the use of
zebrafish technology).9 The latter approach is attractive
because it is close to the animal model and is a good way to
evaluate the therapeutic potential of small molecules at an
early stage. In particular, natural products having macro-
cyclic architectures are attractive due to several reasons: (i)
the macrocyclic shapes represent preorganization, (ii) the
potential to map a large surface area, and (iii) numerous
binding sites.10 Despite all these attractive properties that
are commonly associated with macrocyclic compounds,
building a chemical toolbox having a diverse set of macro-
cyclic compounds is still in its infancy.11 With this objec-
tive, we launched a program that aims to obtain different
types of macrocyclic compounds that could be derived
from carbohydrates as a cheap source for chirality.
Scheme 1. Our Approach To Obtain 14-Membered Macrocycles
Herein, we outline our approach with glycopyranosides
as the starting material (1.1, Scheme 1), which can lead to
accessing various acyclic compounds (see 1.2 and 1.3).12
Through the utilization of functional groups at C-1 and
C-5, we then plan to incorporate amino acid moieties in
two different manners that would lead to two families of
unique macrocyclic glycohybrids(1.4 and 1.5) followingthe
subjection to the “stitching technology”. Our approach
can be general in nature; for example, the use of different
sugars (i.e., glucose, galactose, mannose, etc.) can lead to
producing 14-membered glycohybrids with variation in
their stereochemical display of hydroxyl groups. A specific
example of our approach is also shown in Scheme 1. For
example, if we utilize R-D-glucopyranoside (1.6) as the
starting material, we can aim to access 14-membered,
glycohybrid 1.4a that has retained the stereochemistry of
C-2 to C-5 hydroxyl groups. Inanother case, 14-membered
glycohybrid 1.5a has three hydroxyl groups with retention
ofthe stereochemistry, asitwas in the startingsugar and an
inverted primary hydroxyl group at C-5. Both macrocyclic
compounds 1.4a and 1.5a are planned to be assembled
through the crucial ring-closing metathesis “stitching tech-
nology” on highly functionalized acyclic substrates, 1.7
and 1.8. First, as a proof of concept study, we set our
objective toward achieving the synthesis of two macro-
cyclic targets, 1.4a and 1.5a.
Methyl-R-D-glucopyronoside (2.1, Scheme 2) was used
as the test starting material. Upon subjection to perbenzy-
lation, the pyranoside ring was opened under acidic con-
ditions and then directly applied to reductive alkylation
giving 2.2 in a high yield. The secondary amine was then
coupled with various N-Fmoc protected amino acids,
which upon N-Fmoc removal and amidation resulted in
2.4. At this stage the acyclic precursor was set to bis-
allylation that gave the required bis-allylated product 2.5
needed to test our key stitching technology on this highly
functionalized substrate. To our delight, use of the Grubbs
second generation catalyst13 (G-II, 15 mol %) led to 14-
membered macrocyclic ring formation (olefinic isomeric
ratio not defined yet). Finally, this mixture of two olefinic
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Org. Lett., Vol. 15, No. 3, 2013
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Scheme 2. 14-Membered Macrocycles
Scheme 3. Our Other Synthetic Approach To Obtain 14-Mem-
bered Macrocycles
Figure 1. Zebrafish assay. Angiogenesis: (A) Zoom section of
wild-type or vehicle treated embryo; (B) zoom section of control;
(C and D) zoom sections after treatment with 2.7d, 2.7f, 2.6b, and
2.4b. Early embryo development: (E) DMSO exposed embryos;
(F) small molecule exposed embryos causing a delay in epiboly.
acid chlorides. The next key step in our synthesis was a
Mitsunobu reaction, and this was successfully carried out
using DIAD, PPh3, and DPPA to obtain 3.3 with the
inverted stereochemistry. The corresponding azide 3.3 was
further reduced using a Staudinger reaction to give a
primary amine, which was then coupled with several
N-Fmoc protected amino acids under HOBT, DIC, and
DIPEA in DMF conditions to give 3.4 in good yields. This
was further converted to 3.5 by treatment with DBU
followed by monoallylation reaction using allylbromide,
triethylamine, and the conversion of a secondary amine to
tertiary amide 3.5 using different acid chlorides. Having
key functionalized acyclic starting material, we were ready
to test our crucial ring-closing metathesis “stitching tech-
nology”. As in the previous case, this went very well using
10 mol % Grubbs second generation catalyst (G-II), and
macrocyclic products 3.6 were nicely obtained. Finally,
treatment to hydrogenation conditions led to the complete
synthesis of our desired target 3.7, and six macrocyclic
compounds were obtained using this approach.
compounds was hydrogenated and this provided the
macrocyclic compound 2.7 cleanly. Eight macrocyclic
compounds were synthesized using this approach. All the
products obtained in this scheme were thoroughly purified
and thencharacterized using HPLC-MS and NMR. Inone
case (2.7e), we successfully obtained the X-ray that further
confirmed the macrocyclic ring assignment.
Our synthesis plan to obtain glycol-based 14-membered
macrocyclic compounds, 3.7, is shownin Scheme 3. 3.1 was
obtained from 2.1 in three steps as follows: (i) perbenzyla-
tion, (ii) acid treatment, and (iii) reductive alkylation. This
amine was then converted into amide 3.2 using different
The next plan was to subject our glycohybrid macro-
cyclic collection (78 compounds in total) to various
zebrafish screens to search for functional small molecules.
These screens were related to identify functional chemical
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Org. Lett., Vol. 15, No. 3, 2013

probes affecting epiboly during early embryonic develop-
ment,14 angiogenesis,15 and neurogenesis16 in zebrafish
embryo assays. All three assays are well-documented in
the literature,17 and the detailed procedure is provided in
the Supporting Information. Thus, we identified two novel
glycohybrid 14-membered ring-derived compounds, 2.7d
and 2.7f (Figure 1), as potent inhibitors of trunk angiogen-
esis (i.e., stopped complete angiogenesis at 2.5 μM). In
addtion to this, two more compounds (e.g., an acyclic
derivative, 2.4b, and a 14-membered ring macrocyclic
compound, 2.6b) exihibited partial inhibition at 2.5 μM
and complete inhibition at 5.0 μM. It is too early to predict
the mode of action of these two potent macrocyclic com-
pounds that are structurally closely related. Further work
would be needed to gain deeper insight leading to a better
understanding of the mechanism of action.
In another study examining the effect of small molecules
on epiboly cell movements during early embryonic devel-
opment (Figure 1), we identified two active compounds
(2.7f and S5d). The delay in epiboly was clearly seen in
exposed embryos (i.e., complete inhibition by 2.7f at
2.5 μM and by S5d at 5.0 μM). It was interesting to note
that small molecule 2.7f was also observed to be a potent
inhibitor of angiogenesis.
To summarize, we report here a practical approach to
access functionalized carbohydrate-derived macrocyclic
compounds. The synthesis plan is highly modular and
allows us to obtain a different set of 14-membered macro-
cyclic derivatives. Yet, in our present study, we only
utilized R-D-glucopyranoside as the starting sugar; the
scope of this methodology with other pyranoside deriva-
tives remains to be tested. Using our chemical toolbox
obtainedtodate, itwasfurthertestedina zebrafishembryo
angiogenesis study. This led to identification of two fully
deprotected 14-membered macrocyclic compounds (2.7d
and 2.7f) as potent inhibitors of angiogenesis (i.e., com-
plete inhibition at 2.5 μM). Interestingly, both of these
compoundsare structurally related. One of them(2.7f) also
exhibited complete inhibition of early embryonic develop-
ment in a zebrafish screen at 2.5 μM. In addition to 2.7f,
S5d also exhibited complete inhibition of early embryo-
nic development at 5.0 μM. The discovery of macro-
cyclic compound 2.7f showing two effects is intriguing,
and further work is required to gain a better under-
standing to determine any commonalities regarding the
biological effect.
Acknowledgment. This work was supported by DST
(SR/S1/OC-30/2010) and DBT (102/IFD/SAN/PR 2862/
2010-11) grants to P.A. B.D. and J.S. thank the CSIR
funding agency for the award of the PhD fellowship.
We also thank our analytical team for providing excellent
HPLC-MS and NMR support.
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Supporting Information Available. The detailed syn-
thetic procedure and the analytical data for all the new
compounds are provided. This material is available free
of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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