A modular approach to build macrocyclic diversity in aminoindoline scaffolds identifies antiangiogenesis agents from a zebrafish assay

Article abstract of DOI:10.1002/ejoc.201300409

A modular approach to explore the macrocyclic chemical space around an aminoindoline scaffold is developed. This is achieved by incorporating an amino acid moiety and subsequent stitching technology . Through screening of a zebrafish assay, several antiangiogenesis agents are identified. A modular approach to explore the macrocyclic chemical space around an aminoindoline scaffold is developed. This is achieved by incorporating an amino acid moiety and subsequent stitching technology . Through screening of a zebrafish assay, several antiangiogenesis agents are identified. Copyright

Full text of DOI:10.1002/ejoc.201300409

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201300409
A Modular Approach to Build Macrocyclic Diversity in Aminoindoline
Scaffolds Identifies Antiangiogenesis Agents from a Zebrafish Assay
Srinivas Chamakuri,[a] Shiva Krishna Reddy Guduru,[a] Sreedhar Pamu,[a][‡]
Gayathri Chandrasekar,[b] Satish Srinivas Kitambi,*[b,c,d] and Prabhat Arya*[a]
Keywords: Medicinal chemistry / Angiogenesis / Chemical probes / Molecular diversity / Nitrogen heterocycles /
Macrocycles
A modular approach to explore the macrocyclic chemical
space around an aminoindoline scaffold is developed. This is
achieved by incorporating an amino acid moiety and subse-
quent “stitching technology”. Through screening of a zebra-
fish assay, several antiangiogenesis agents are identified.
of undertaking biological targets that are beyond our reach
at present.[15,7]
Introduction
With the goal of expanding the currently accepted chemi-
cal space to search for small molecule modulators of pro-
tein–protein[1–3] and DNA/RNA–protein[4] interactions and
signaling pathways,[5,6] the past few years have seen a grow-
ing interest in accessing small molecules[7–9] that are more
natural product like, and many of these small molecules are
obtained through the inspiration of bioactive natural prod-
ucts.[10–12] Traditionally, small molecules that are broadly
available to the medicinal chemistry community for early
biological evaluation are rich in sp2 character, and in gene-
ral, they lack the features that are commonly found in bio-
active natural products.[13,14] Several leading review articles
have been written on this topic, and they challenge the or-
ganic synthesis community to access small molecules for
biological evaluation in the broad areas of protein–protein
interactions and other bio-macromolecular interac-
tions.[14,7,10,12] It is hoped that by going beyond the conven-
tional chemical space and by enriching our small molecule
collection to be more natural product like, we will identify
functional small molecules for areas that are considered dif-
ficult.[15,7] Moreover, this will allow us to expand the scope
In particular, the interest in accessing functionalized
large ring derivatives (i.e., macrocyclic compounds) is grow-
ing due to several reasons: (1) Medium-to-large ring struc-
tures present an opportunity to map a large surface area to
interact with protein targets. (2) Preorganization in large
rings allows several functional groups in specific orienta-
tions to be displayed. (3) In general, cyclic compounds pos-
sess better cell permeation properties.[16] Despite all of these
attractive features that large rings offer and despite the ex-
cellent track record of several macrocyclic natural products
as drugs, the full potential of this area remains to be ex-
plored thoroughly.[16–22] One of the challenges in this arena
is the development of synthesis methods that are modular
and practical in nature and that would allow different types
of functionalized medium to large rings to be obtained.
A few years ago, we initiated a synthesis program that
was aimed at developing methods to allow us access to
functionalized, enantioenriched natural product inspired
scaffolds.[23–25] In one study,[23] we developed a practical
synthesis of an aminoindoline scaffold having three orthog-
onally protected functional groups. In this report, we dem-
onstrate that this bicyclic scaffold can be converted into a
tricyclic system through an in situ, stereocontrolled aza-
Michael reaction. Our approach is general and can be
transferred to the solid phase, which allowed us to obtain
a proof of concept library for further biological evaluation.
[a] Dr. Reddy’s Institute of Life Sciences, University of Hyderabad
Campus,
Gachibowli, Hyderabad 500046, India
E-mail: prabhata@drils.org
Homepage: www.prabhatarya.org
[b] School of Life Sciences, Södertörns Högskola,
14189 Huddinge, Sweden
[c] Department of Biosciences and Medical Nutrition, Karolinska
Institutet,
Results and Discussion
17177 Stockholm, Sweden
[d] Division of Molecular Neurobiology, Department of Medical
Biochemistry and Biophysics, Karolinska Institutet,
17177 Stockholm, Sweden
As an extension of this work, with the objective to ex-
plore macrocyclic chemical space,[26,27] we report herein a
modular method that allowed us to incorporate different
types of medium/large ring skeletons (see 1.2, 1.3, and 1.4;
Scheme 1) into enantioenriched aminoindoline scaffold 1.1.
E-mail: satish.kitambi@ki.se
[‡] Current address: McGill University, Montreal
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300409
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SHORT COMMUNICATION
Scheme 1. The proposed plan to obtain macrocyclic-derived compounds 1.2, 1.3, and 1.4 from enantioenriched aminoindoline scaffold
1.1. P1: NHTeoc; P2: OBz; P3: NHAlloc.
There are several attractive features of aminoindoline scaf- riched aminoindoline substructure, a trans-fused 12-mem-
fold 1.1 that make it an attractive starting point to incorpo- bered ring, and the presence of an amino acid functionality
rate various medium/large rings into the skeleton. These in- in the macrocyclic skeleton to allow variation in the nature
clude the presence of (1) a trans-2,3-amino alcohol moiety of the chiral side chain. Similarly, target 1.3 provides the
that can further be utilized to build trans-fused 12-mem- aminoindoline substructure and an additional 11-mem-
bered ring 1.2, (2) a 1,2-amino alcohol that utilizes the bered ring having an amino acid in the skeleton to allow
indoline nitrogen atom to provide 11-membered ring 1.3, variation in the chiral side chain. Finally, our third target,
and (3) 1,3-orthogonally protected diamino functional that is, compound 1.4, contains an additional bridged 11-
groups that can lead to bridged 11-membered ring 1.4. In memebred ring that utilizes both nitrogen atoms of an
all three strategies, we utilized a common approach to in- aminoindoline scaffold. Altogether, these three divergent
corporate the amino acid functionality, and ring-closing strategies with a common objective provide an excellent op-
metathesis[28–30] was used as stitching technology to con- portunity to build a unique chemical toolbox having com-
struct the macrocyclic ring. For example, final target 1.2 pounds that will represent the privileged substructure and
presents several interesting features, including an enantioen- additional different types of large-ring derivatives to explore
Scheme 2. The synthesis of 1.2 having a 12-membered macrocyclic ring on an aminoindoline scaffold. Py = pyridine, Bz = benzoyl, DBU
= 1,8-diazabicyclo[5.4.0]undec-7-ene, TBAI = tetrabutylammonium iodide.
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A Modular Approach to Build the Macrocyclic Diversity
their value in biology as modulators of protein–protein in- completing the synthesis of macrocyclic products 1.2. All
teractions and signaling pathways.
products were thoroughly purified and characterized (see
Our synthesis method to obtain 12-membered ring the Supporting Information).
macrocyclic ring derivative 1.2 from an enantioenriched
Our next plan was to develop a synthesis method to ac-
aminoindoline scaffold is outlined in Scheme 2. As we re- cess various macrocyclic compounds from 1.3 (Scheme 3).
ported the synthesis of an aminoindoline scaffold earlier,[23] To achieve this objective, we again utilized compound 2.2
a slightly revised procedure gave us easy access to 2.2 from as the starting material. The direct coupling with various
2.1 (note: the detailed synthesis procedure is provided in the amino acid chlorides (i.e., to obtain the first diversity point
Supporting Information).
as R1) was achieved. The Fmoc group was removed, and
The free amine was acylated (i.e., R4 as the first diversity the amine was acylated to add the second diversity point as
point), and following removal of the benzyloxycarbonyl R2. Because we planned to utilize the aminoindoline side
(Cbz) group (10% Pd over C), it was then subjected to re- chain oxygen atom to obtain our macrocyclic ring, the
ductive alkylation to obtain secondary amine 2.3 in good indoline amine was converted into a tertiary amide follow-
yield (i.e., R3 as the second diversity point). The secondary ing removal of the Cbz group to give us two additional
amine was then coupled with different 9-fluorenylmethoxy- diversity sites (R3 and R4). As described earlier, allylation
carbonyl (Fmoc) amino acids that allowed us to introduce of 3.2 was performed, and as in the previous case, under
the diversity site as R1 (i.e., the side chain of an amino the reaction conditions 3.2 was also debenzoylated, which
acid). The Fmoc group was removed, and the free amine led to O-allylation at the primary hydroxy group. The bisal-
was then acylated with acid chlorides to provide 2.4 with lylated product was then subjected to ring-closing metathe-
the fourth diversity point (R2). Upon treatment under basic sis by using Grubbs chemistry. Once again, this reaction
conditions with allyl bromide, bisallylated product 2.5 was worked well and the 11-membered cyclic ring was easily ob-
produced in respectable yield. Under reactions conditions tained. To validate the generality of our approach, we tried
that may have trace amounts of moisture, hydrolysis of the this method in four cases and all of them successfully pro-
benzoyl ester was also observed, which was then followed duced the macrocyclic products. We subjected all four
by O-allylation. The next crucial step was to explore the macrocycles to double bond removal under hydrogenation
ring-closing metathesis based stitching technology on conditions, and their corresponding saturated derivatives
highly functionalized substrate 2.5. This reaction worked were easily obtained as 1.3. All the products were thor-
well, and as we proposed, the 12-membered macrocyclic oughly purified and characterized by HPLC–MS and NMR
ring was easily obtained. The cyclic product was then sub- spectroscopy.
jected to hydrogenation to obtain the saturated version as
Our final approach to develop a method to access 11-
product 1.2, which was characterized through various ana- membered bridged macrocyclic derivatives (see, 1.4) is
lytical techniques (HPLC–MS and NMR spectroscopy). shown in Scheme 4. As in the previous case, we obtained
Our approach is general in nature, and this was tested by 4.1 from 3.1 in easy steps. At this stage, the amino group
Scheme 3. Synthesis of 1.3 with an 11-membered ring. DIPEA = N,N-diisopropylethylamine.
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S. S. Kitambi, P. Arya et al.
SHORT COMMUNICATION
Scheme 4. The incorporation of the 11-membered bridged macrocycle.
on scaffold 3.1 was deprotected and further acylated to ob- mented in the literature[33,35] and are routinely utilized in
tain 4.1. Following the conversion of the side chain hydroxy our laboratories. A major advantage with the use of the
group from OBz to OMe or OBn, the substrate was sub- zebrafish technology is that it is close to the in vivo model,
jected to the bisallylation reaction conditions. The bisallyl- and it is a cheap way to evaluate compounds in a high-
ated product was then subjected to crucial stitching technol- throughput manner. Details of the screening procedure are
ogy by using ring-closing metathesis. To our pleasant sur- provided in the Supporting Information. The zebrafish
prise, this reaction worked well, and final product 1.4 ob- screen identified 3.1b, 3.2b, and S14 (Figure 1) as antiangi-
tained after double bond removal, and it was thoroughly ogenesis agents with partial inhibition at 2.5 μm and com-
purified and characterized (see the Supporting Infor- plete inhibition at 5.0 μm. Compounds 3.1d, 2.3c, and 2.3d
mation). Once again, to explore the validity of this method, were found to exhibit complete inhibition at 2.5 μm. In an-
we examined two other substrates, and our approach to other screen of early embryonic development, we identified
build the 11-membered bridged macrocyclic ring worked in S26 functioning at 3.0 μm.
both cases. Careful analysis of the 3D model of 1.4a indi-
cated that the benzylic nitrogen atom occupies a pseudoax-
ial position and this brings the CH2OMe group out of the
plane due to a relative 1,2-trans orientation. This may be a
Conclusions
crucial factor in bringing the two olefinic groups in proxim-
With the goal of building a diverse set of small molecules
ity to facilitate formation of the 11-membered bridged having a privileged substructure and different types of me-
macrocycle. To the best of our knowledge, this strategy to dium to large ring derivatives, we developed a diversity-
obtain different macrocyclic rings attached to the amino- based method. The first study utilized the primary hydroxy
indoline scaffold is unique and has not been reported. It on the side chain and the benzylic amine to build a 12-
also provides an excellent opportunity to build a novel, nat- membered macrocyclic ring. Using a similar strategy and
ural product inspired chemical toolbox to explore its value through the use of the primary hydroxy group and the
in a search of functional small molecules.
indoline amino group, an 11-membered ring was synthe-
All the small molecules obtained from this project (in sized. Finally, the use of both nitrogen atoms (i.e., primary
total 39 compounds) were then subjected to an embryo ze- and secondary amines) led to the successful synthesis of an
brafish screen in three different assays: angiogenesis,[31–33] 11-membered bridged macrocycle. Upon evaluation of our
early embryonic development,[34] and neurogenesis.[33,35]
A
small molecules in a zebrafish assay, we identified three
detailed list of all compounds tested in this study is shown novel compounds as antiangiogenesis agents. These find-
in the Supporting Information. These assays are well docu- ings are at an early stage and further work is required to
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A Modular Approach to Build the Macrocyclic Diversity
gain deeper insight into the mode of action of these func-
tional small molecules.
Supporting Information (see footnote on the first page of this arti-
cle): General methods, experimental procedures, spectral data, and
zebrafish screening.
Acknowledgments
This work was supported by the Department of Science and Tech-
nology (DST), New Delhi (DST and DBT grants to P. A.). S. C.
and S. K. R. G. thank the Council of Scientific and Industrial Re-
search (CSIR), New Delhi for the award of Ph.D. fellowships. The
authors also thank the ILS analytical team for providing excellent
HPLC–MS and NMR support. S. K. thanks Södertörns Högskola
for the financial support to perform the zebrafish assays.
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Received: March 18, 2013
Published Online: May 21, 2013
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