Tetrahydroquinoline-derived macrocyclic toolbox: The discovery of antiangiogenesis agents in zebrafish assay

Article abstract of DOI:10.1021/ml400026n

A novel approach to incorporate the macrocyclic rings onto the privileged substructure, i.e., tetrahydroquinoline scaffold, is developed. The presence of an amino acid-derived moiety in the macrocyclic skeleton provides an opportunity to modulate the nature of the chiral side chain. Further, evaluation in a zebrafish screen identified three active small molecules (2.5b, 3.2d, and 4.2) as antiangiogenesis agents at 2.5 μM.

Full text of DOI:10.1021/ml400026n

Letter
pubs.acs.org/acsmedchemlett
Tetrahydroquinoline-Derived Macrocyclic Toolbox: The Discovery of
Antiangiogenesis Agents in Zebrafish Assay
Shiva Krishna Reddy Guduru,† Srinivas Chamakuri,† Gayathri Chandrasekar,‡ Satish Srinivas Kitambi,*,‡,§
and Prabhat Arya*,†
†Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
‡School of Life Sciences, Sodertorns Hogskola, Sweden
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§Division of Molecular Neurobiology and Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Sweden
S
* Supporting Information
ABSTRACT: A novel approach to incorporate the macro-
cyclic rings onto the privileged substructure, i.e., tetrahy-
droquinoline scaffold, is developed. The presence of an amino
acid-derived moiety in the macrocyclic skeleton provides an
opportunity to modulate the nature of the chiral side chain.
Further, evaluation in a zebrafish screen identified three active
small molecules (2.5b, 3.2d, and 4.2) as antiangiogenesis
agents at 2.5 μM.
KEYWORDS: Natural product-inspired compounds, macrocyclic diversity, zebrafish screen, antiangiogenesis agents
he growing quest to undertake biological targets that
accessing different-types of functionalized large ring com-
pounds. In one study, we were interested in utilizing an
enantioenriched tetrahydroquinoline scaffold, (1.1, Scheme 1)
that was reported by us earlier.23−27 The presence of a β-amino
acid functionality and three contiguous chiral functional groups
are the two attractive features of this scaffold. Our synthesis is
practical and enantioselective in nature and allows us to access
this scaffold in sufficient quantities in a short period that
T
belong to the family of protein−protein1,2 and DNA/
RNA−protein3 interactions in the signaling pathway arena4,5 is
challenging the current thinking in going beyond the
conventional chemical space to search for functional small
molecules. Typically small molecules in the drug discovery
research are rich in sp2 character,6,7 whereas natural products8
that have an excellent track record in the domain of
biomacromolecular interactions are relatively complex in nature
and present a diverse array of chiral functional groups. In
particular, macrocyclic natural products9 are proven to exhibit
remarkable biological responses when it comes to modulating
ppi through small molecules. There are several factors
associated with macrocyclic natural product derivatives, and
these include (i) an ability to map a large surface area, (ii)
numerous binding interaction options, (iii) enhanced cell
permeation properties when compared to their liner derivatives,
and (iv) the dynamic preorganized structures to display various
functional groups. Despite all these benefits that are associated
with bioactive macrocyclic natural products, we have not seen a
significant growth in building a chemical toolbox having diverse
sets of different-types of macrocyclic shapes available to explore
their biological value.9−15 Because of the inherited challenges
associated with complex bioactive natural products (i.e.,
macrocyclic or nonmacrocyclic compounds), the interest in
building a chemical tool box having small molecules that are
obtained by inspirational approaches is also rising.16−22
Scheme 1. Incorporation of Different Macrocyclic Rings
onto an Enantioenriched Tetrahydroquinoline Scaffold
Received: January 17, 2013
Accepted: May 24, 2013
Toward this objective, a few years ago, we embarked a
program in developing several novel approaches to allow
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
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utilized a stereoselective aza Michael reaction as the key step.
To explore further the large ring-based chemical space around
this scaffold, we report here our approach to build further the
functionalized 12- and 14-membered rings onto this scaffold
(see, 1.2−1.4).
Scheme 3. Synthesis Route to Obtain a 12-Membered
Macrocyclic Ring onto the Tetrahydroquinoline Ring That
Utilizes Amino Alcohols
There are two main objectives in our design strategy, first is
to retain the functionalized privileged substructure, i.e.,
tetrahydroquinloine, and second is to map the macrocyclic
chemical space with the additional functional groups. For
example, target 1.2 has the additional 12-membered ring with
an incorporation of an amino acid moiety in the skeleton. The
ring-closing metathesis reaction was utilized as the stitching
technology to obtain the macrocyclic rings. Using a similar
approach, one can also obtain compound 1.3 with a 12-
membered ring having the connectivity through the ether
linkage. In addition to these two compounds, we also plan to
incorporate 14-membered macrocyclic ring onto the tetrahy-
dorquinoline scaffold having an amino acid moiety in the ring
skeleton. Overall, our approach to building different types of
large ring skeletons onto the tetrahydroquinoline scaffold
provides an excellent opportunity to accessing a chemical tool
box with a diverse set of functionalized large ring-based
derivatives.
the free carboxylic acids, and this successfully led to the
synthesis of 4 examples (3.4a−d). Once again, the products are
obtained as a single isomer and the olefin geometry is not
defined yet. Our plans to incorporate a 14-membered ring onto
the tetrahydroquinoline scaffold are shown in Scheme 4.
Our synthesis approach to incorporate a 12-membered ring
onto an enantioenriched tetrahydroquinoline scaffold is shown
in Scheme 2. As reported by us earlier,28 we obtained an
Scheme 4. Synthesis Route to Incorporate a 14-Membered
Ring onto an Enantioenriched Tetrahydroquinoline Scaffold
Scheme 2. Synthesis Route to Obtain a 12-Membered
Macrocycle onto the Tetrahydroquinoline Ring
enantioenriched compound 2.2 from 2.1 in a number of well-
established steps. One of the key advantages with this scaffold is
that it can be accessed in several gms quantities in a short
duration. The presence of the β-amino acid moiety in this
scaffold is an attractive feature and is a subject of further
modification. For example, the free carboxylic acid group (2.2)
was coupled with several amino esters to obtain compound 2.3
(R1 as the diversity site). This was easily converted to bis-allyl
derivative 2.4 needed for building the 12-membered macro-
cyclic ring. Upon subjection to the ring-closing metathesis
stitching technology,29−31 we successfully obtained the cyclic
product 2.5 in good yields as the single isomer (olefin geometry
is not defind yet). Our approach is general in nature, and as a
proof of concept studies, four macrcocyclic compounds 2.5a−d
were obtained. All the products in this scheme are thoroughly
purified and characterized using HPLC-MS and NMR. The
detailed procedure is provided in the Supporting Information.
In another similar approach, we utilized the corresponding
amino-alcohol derivatives 3.1 (see Scheme 3) to couple with
Compound 4.1 as the starting material (see Supporting
Information) was utilized to obtain 4.2 in two steps that
followed the carboxyl ester reduction and aromatic amine
protection as −NAlloc. Following the oxidation of a primary
hydroxyl group, it was then reductively alkylated to obtain the
secondary amine (with the first diversity as R3) and then
coupled with several amino acids and further amidation
(second and third diversity sites, R1 and R2) to obtain 4.4.
This was then subjected to N-allylation followed by ring closing
metathesis stitching technology using second generation
Grubbs catalyst giving the 14-membered ring-derived com-
pounds 4.5. This reaction worked well and was also utilized to
obtain four test macrocyclic compounds 4.5a−d. All the
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products were purified over silica gel and thoroughly
characterized by HPLC-MS and NMR (note: olefin geometry
is not defined yet). As test studies, in two cases, the acetonide
protection was removed under mild acidic conditions giving the
macrocyclic compounds with two free hydroxyl groups (see
4.6).
Having a chemical toolbox available to explore its biological
value, we then decided to search for functional small molecules
in three zebrafish screens,32,33 and these are (i) angio-
genesis,34,35 (ii) an early embryonic development,35 and (iii)
neurogenesis.36 These assays are well-established in our lab and
utilize the procedure that is thoroughly documented in the
literature.37,38 The detailed procedure is also provided in the
Supporting Information. Of all the compounds tested from this
toolbox (60 compounds in total), we identified three
compounds (2.5b, 3.2d, and 4.2) that exhibited the inhibition
of angiogenesis at 2.5 μM. These results are shown in Figure 1.
Figure 2. Zebrafish screen for an early embryo development: (A)
DMSO exposed embryos at 10 hpf of development and (B) small
molecule 2.5c exposed embryos causing a delay in epiboly. One
macrocyclic derivative (2.5c) and two tetrahydroquinoline-based
compounds (S8d and 4.2) exhibited the complete inhibition of an
early embryo development at 2.5 μM.
in the Supporting Information). This may indicate that the
minimum concentration required to produce any significant
effect on these biological processes is above 2.5 μM. The severe
effect was seen as the complete inhibition of angiogenesis and
epiboly, and the partial inhibition was characterized by the
inhibition of angiogenesis of more than 50% of vessels.
It is interesting to note that the functional macrocyclic
compounds (2.5b and 2.5c) in both assays are structurally
related. It would be excellent to find the exact mechanism of
action of these compounds and to determine if there is any
common mode of action in these two phenotype experiments.
With an objective to incorporate different macrocyclic rings
onto the tetrahydroquinoline scaffold, we successfully devel-
oped several approaches. The presence of the privileged
substructure and the additional macrocyclic rings (for example,
functionalized 12- and 14-membered rings) are two unique
features in our design strategy. Further, the incorporation of an
amino acid in the large ring skeleton allows an opportunity to
modulate the nature of the side chain (for example, chiral polar
to nonpolar groups through utilizing natural and un-natural
amino acids). Finally, when tested this tool box in a zebrafish
screen, we identified three functional small molecules active as
antiangiogenesis agents at 2.5 μM and three as inhibitors of an
early embryonic development. To understand the precise mode
of action of these compounds that are active in phenotypic
screens, much work would be needed. This will be reported as
these findings become available.
Figure 1. Zebrafish screen for angiogenesis: (A) zoom section of wild-
type or vehicle treated embryo and (B,C) zoom sections after
treatment with compound 2.5b. One macrocyclic derivative (2.5b)
and two tetrahydroquinoline-based compounds (3.2d and 4.2)
showed complete inhibition at 2.5 μM.
In another zebrafish screen to search for functional small
molecules affecting an early embryonic development (see
Figure 2), we identified three compounds (2.5c, S8d, and 4.2)
that inhibited at 2.5 μM.
In our zebrafish studies, we exposed a total of 30 embryos
per compound producing defects in angiogenesis and early
embryo developmental stage. We then quantified the number
of embryos exhibiting severe defects in each treatment.
Embryos at 2.5 μM completely exhibited severe phenotype,
and this percentage dropped drastically when the concentration
was slightly lowered (note: the detailed information is provided
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed synthesis procedure along with the analytical data for
all new compounds and the zebrafish screen. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Letter
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AUTHOR INFORMATION
Corresponding Author
*(P.A.) E-mail: prabhata@drils.org.
Author Contributions
The manuscript was written through contributions of all
authors.
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Funding
This work was supported by DST (SR/S1/OC-30/2010) and
DBT (102/IFD/SAN/PR-2863) grants to P.A.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
S.K.R.G. and S.C. thank CSIR funding agency for the award of
the Ph.D. fellowships. We also thank the ILS analytical team for
providing an excellent HPLC-MS and NMR support. S.S.K.
thanks Sodertorns Hogskola for the financial support to
perform zebrafish assays.
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