Diversity through a branched reaction pathway: Generation of multicyclic scaffolds and identification of antimigratory agents

Article abstract of DOI:10.1002/chem.201002195

A library of 91 heterocyclic compounds composed of 16 distinct scaffolds has been synthesized through a sequence of phosphine-catalyzed ring-forming reactions, Tebbe reactions, Diels-Alder reactions, and, in some cases, hydrolysis. This effort in diversity-oriented synthesis produced a collection of compounds that exhibited high levels of structural variation both in terms of stereochemistry and the range of scaffolds represented. A simple but powerful sequence of reactions thus led to a high-diversity library of relatively modest size with which to explore biologically relevant regions of chemical space. From this library, several molecules were identified that inhibit the migration and invasion of breast cancer cells and may serve as leads for the development of antimetastatic agents.

Full text of DOI:10.1002/chem.201002195

FULL PAPER
DOI: 10.1002/chem.201002195
Diversity Through a Branched Reaction Pathway: Generation of Multicyclic
Scaffolds and Identification of Antimigratory Agents
Zhiming Wang,[a, c] Sabrina Castellano,[a] Sape S. Kinderman,[a] Christian E. Argueta,[b]
Anwar B. Beshir,[b] Gabriel Fenteany,*[b] and Ohyun Kwon*[a]
Abstract: A library of 91 heterocyclic
compounds composed of 16 distinct
scaffolds has been synthesized through
terms of stereochemistry and the range
of scaffolds represented. A simple but
powerful sequence of reactions thus
led to a high-diversity library of rela-
tively modest size with which to ex-
plore biologically relevant regions of
chemical space. From this library, sev-
eral molecules were identified that in-
hibit the migration and invasion of
breast cancer cells and may serve as
leads for the development of antimeta-
static agents.
a
sequence of phosphine-catalyzed
ring-forming reactions, Tebbe reactions,
Diels–Alder reactions, and, in some
cases, hydrolysis. This effort in diversi-
ty-oriented synthesis produced a collec-
tion of compounds that exhibited high
levels of structural variation both in
Keywords: antimigratory agents
·
chemical biology · diversity-orient-
ed synthesis · heterocycles · synthe-
sis design
Introduction
function.[1] If we had such insight, designing small molecule
modulators of proteins of known structures (crystallographi-
cally or spectroscopically) would be relatively easy. Our sci-
entific understanding today is, however, quite far from al-
lowing us to develop desired functions based on structure
alone—without a priori information. One approach toward
broadening our understanding of the relationship between
structure and function would be to generate many new
small molecules that modulate proteinsꢀ functions and study
the interactions between them.
At its heart, modern chemistry is a quest to understand the
relationship between structure and (biological or material)
function. From a biological point of view, we do not yet un-
derstand the rules for predicting 1) whether a particular
small molecule will interact with proteins, 2) what protein it
will interact with if it does, and 3) which mode of interaction
it will employ. Chemists have formulated only relatively lim-
ited empirical rules (such as Lipinskiꢀs “rule of five”) to
guide efforts to generate molecules of desirable biological
In this context, collections of compound libraries have
been established as common starting points for the study of
chemical genetics and the discovery of new drugs.[2] The de-
velopment of efficient methods for the construction of libra-
ries encompassing the maximum amount of chemical space
is a particularly challenging task for organic chemists.[3] Es-
tablishing the maximum amount of skeletal diversity is the
key factor toward improving the efficiency for novel thera-
peutic leads screening.[4] Diversity-oriented synthesis (DOS)
entails the development of pathways leading to the efficient
synthesis of collections of small molecules exhibiting rich
skeletal and stereochemical diversity.[5] Compared with li-
braries constructed from common scaffolds decorated with
diverse substituents,[2b,6] examples of libraries of small mole-
cules featuring high degrees of skeletal diversity are rela-
tively limited.[7] In addition, although such exercises are un-
dertaken based on the premise that a diverse range of scaf-
folds should provide a higher chance for discovery of small-
[a] Dr. Z. Wang, Dr. S. Castellano, Dr. S. S. Kinderman, Prof. O. Kwon
Department of Chemistry and Biochemistry
University of California, Los Angeles
607 Charles E. Young Drive East, Los Angeles
CA 90095-1569 (USA)
Fax : (+1)310-206-3722
E-mail: ohyun@chem.ucla.edu
[b] C. E. Argueta, Dr. A. B. Beshir, Prof. G. Fenteany
Department of Chemistry, University of Connecticut
55 North Eagleville Road, Storrs, CT 06269-3060 (USA)
Fax : (+1)860-486-2981
E-mail: gabriel.fenteany@uconn.edu
[c] Dr. Z. Wang
School of Chemical Engineering, Changzhou University
Changzhou 213164, Jiangsu, (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002195.
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649

molecule biological functional modulators, its actual realiza-
tion is rarely reported. Herein, we report a highly efficient
and modular synthesis of a library of 91 multicyclic hetero-
cycles with 16 distinctive scaffolds and the resulting identifi-
cation of new antimigratory agents.
Results and Discussion
Cycloaddition of electron-deficient allenes under phosphine
catalysis is a robust method for obtaining a variety of carbo-
and heterocycles in an atom-economical and environmental-
ly friendly manner.[8] The phosphine-catalyzed reactions are
typically high yielding and compatible with solid phase syn-
thetic processes. For example, using methodology developed
by Luꢀs group[9] and our own,[10] we have previously generat-
ed a library of small-molecule protein geranylgeranyltrans-
ferase type I (GGTase-I) inhibitors through solid-phase
split-pool synthesis.[6d,11] The pyrrolines and tetrahydropyri-
dines resulting from the phosphine catalysis of allenoates
and imines possess a common a,b-unsaturated ester group.
In our previous synthesis of GGTase-I inhibitors, the a,b-
enoate functionality provided a handle for highly diastereo-
selective Michael additions of thiols. Although the conjugate
addition of thiols provided only a relatively moderate level
of skeletal variation, the pentasubstituted pyrrolidine prod-
ucts were identified as the first small-molecule RabGGTase-
specific inhibitors.[11–12] We envisioned that the versatile
chemistry of conjugated enoates might provide a handle for
further scaffold diversification. When the C=O moiety of an
a,b-unsaturated ester is methylenated, the resulting dienol
ether (an electron-rich diene) can undergo Diels–Alder re-
actions with electron-deficient dienophiles to generate a va-
riety of fused heterocyclic compounds possessing distinctive
frameworks (Scheme 1).
Scheme 1. Branched pathway for the construction of a library of 16 dis-
tinctive scaffolds; a) and b) phosphine-catalyzed ring-forming reactions;
c) Tebbe reactions, d) Diels–Alder reactions. Reaction conditions:
a) PBu3 (20 mol%), benzene, RT, 12 h; b) PBu3 (20 mol%), CH2Cl2, RT,
12 h; c) Tebbe reagent (3 equiv), pyridine (30 mol%), THF, RT, 12 h;
d) for 7a–7i, maleimide (4 equiv), MeOH (5–15%), CH2Cl2, RT, 48 h;
for 8a–8j, N-phenyltriazolinedione (2 equiv), CH2Cl2, 08C, 1 h; for 9a–
9g, tetracyanoethylene (2 equiv), CH2Cl2, 08C, 1 h; for 10a–10d, dichlor-
obenzoquinone (4 equiv), toluene, 808C, 5 h; for 10e, benzoquinone
(4 equiv), toluene, 808C, 5 h; then CHCl3, SiO2, Et3N, RT, 12 h; for 11a–
11 f, maleimide (4 equiv), MeOH (5–15%), CH2Cl2, RT, 48 h; for 12a–
12e, N-phenyltriazolinedione (2 equiv), CH2Cl2, À788C, 5 h; for 13a–13 f,
tetracyanoethylene (2 equiv), CH2Cl2, À788C, 5 h; for 14a–14c, benzo-
quinone (4 equiv), toluene, 808C, 10 h; for 15a–15j, imine (4 equiv), tolu-
ene, 658C, 24 h. See the Supporting Information for the list of the dieno-
philes tested, the reaction details, and the compound structures.
To begin exploring this concept, we prepared, as starting
materials, the pyrrolines 3a–3j and the tetrahydropyridines
4a–4 f, through phosphine-catalyzed ring-forming reactions
between the allenoates 1 and the imines 2, in excellent
yields (90–97%) on multigram scales (Scheme 1).[9–10] The
methylenation of the a,b-unsaturated esters with the Tebbe
reagent[13] gave the corresponding ethoxy dienes 5a–5j and
6a–6 f with good reaction efficiencies (50–82%). Although
these enol ethers are slightly unstable on silica gel or in
CDCl3 solution, some of them (e.g., 5b, 6a, and 6e) could
be stored at À208C for up to one month, without decompo-
sition, after purification. All of the multiply substituted pyr-
rolines 3 and tetrahydropyridines 4 gave the desired prod-
ucts of their Tebbe reactions.
The Diels–Alder reactions between the Tebbe reaction
products and electron-deficient dienophiles provided anoth-
er key skeleton-diversifying branch in our DOS pathway
(Scheme 2). Through dienophile screening, we identified
maleimides, N-phenyl triazolinedione, tetracyanoethylene,
imines, benzoquinone, and 2,6-dichlorobenzoquinone as
very good reaction substrates for the Diels–Alder reactions
of the ethoxy dienes. Although the reaction yields were only
Scheme 2. Structures of 16 dienes and 12 dienophiles. Bn=benzyl; Bs=
benzenesulfonyl; Ts=tosyl (p-toluenesulfonyl).
moderate to good (35–85%), the reaction stereoselectivities
were excellent. Indeed, considering that the compounds in
the library possess up to six stereogenic centers, the selectiv-
ities of the Diels–Alder reactions are quite remarkable.
Specifically, we detected only single diastereoisomeric
products from the cycloadditions of the pyrroline-derived
dienes 5 with maleimides, N-phenyltriazolinedione, tetracya-
noethylene, and 2,6-dichlorobenzoquinone and from the cy-
cloadditions of the tetrahydropyridine-derived dienes 6 with
N-phenyl triazolinedione, benzoquinone, and imines. The cy-
cloadditions of the dienes 6 with maleimides and tetracyano-
ethylene exhibited diastereoselectivities of up to 6:1 and
10:1, respectively. When benzoquinones were used as the di-
enophiles, we detected no direct Diels–Alder reaction prod-
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Diversity Through a Branched Reaction Pathway
FULL PAPER
ucts; instead, we isolated the oxidized benzoquinones 10a
and 14c and the naphthoquinones 10e and 14a (Scheme 3).
Based on the isolation of dienophile-derived hydroquinone
byproducts, we surmise that dehydrogenation or dehydro-
reactions of the tetrahydropyridine-derived dienes 6 dis-
played relatively mixed facial selectivities. The reaction with
maleimide, although endo-selective, produced the two dia-
stereoisomers 11aa and 11ab in a 2:1 ratio, presumably be-
cause the C2 stereogenic center
was too far removed from the
reaction center to exert a signif-
icant steric bias. The dieno-
philes N-phenyltriazolinedione,
tetracyanoethylene, and benzo-
quinone underwent [4+2] cy-
cloadditions by adding to the
diene 6 from the face opposite
the C2 aryl group (yielding 12a,
13a, and 14c, respectively). In-
terestingly, for imine dieno-
philes, the endo-selective Diels–
Alder reaction provided, for ex-
ample, the octahydro-1,6-naph-
thyridine 15a, in which the
tosyl group in the tetrahydro-
pyridine ring of the diene 6
controlled the facial approach
of the imine dienophile. X-ray
crystallography revealed that
the tetrahydropyridine[10a] and
the
octahydronaphthyridine
featured anti relationships be-
tween their a-phenyl and N-
tosyl groups.
Scheme 3. Representative compounds possessing distinctive scaffolds in the library.
Surprisingly, the Diels–Alder
chlorination occurred, following the Diels–Alder reactions,
in the presence of excesses of benzoquinone dienophiles.[14]
It is also notable that the cyclohexene double bond, result-
ing from the Diels–Alder reaction, isomerized into conjuga-
tion with the benzoquinone in 10a.
reaction products featuring enol ether units were quite
stable during purification through silica gel. Most of them
could be stored at À208C for several months without detect-
able decomposition. The enol ether units were hydrolyzed
to the corresponding ketone products (16–18) upon treat-
ment with a solution of aqueous HCl (0.1m) in acetone
(Scheme 4). Only a single isomer was detected in each case;
Given the complexity of the structures of the library com-
pounds, determining their structures, especially their stereo-
chemistry, was a challenge. The relative configuration of
each scaffold obtained from the Diels–Alder reactions
(Scheme 3), with the exception of 14c, was established un-
equivocally through X-ray crystallographic analysis (see the
Supporting Information). The structure of 14c was estab-
1
lished based on H, 13C, DEPT, and COSY NMR spectro-
scopic and mass spectrometric data. In addition, compound
14c, when left at room temperature for over 6 months, was
converted into its isomer 14c’, which features its enol ether
double bond in conjugation with the benzoquinone motif
(compare with compound 10a); the X-ray diffraction data
of the crystalline solid 14c’ provided the relative configura-
tion of compound 14c. As verified by the X-ray crystallo-
graphic analysis, for the dienes 5, the chiral center(s) created
in the phosphine-catalyzed [3+2] ring-forming reactions
controlled the face from which the dienophile approached
the diene in endo-selective Diels–Alder reactions (7d, 8a,
9a, 10a); specifically, dienophiles approached the dienes 5
from the face opposite the C2 substituent. The Diels–Alder
Scheme 4. Hydrolysis of enol ethers to ketones.
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G. Fenteany, O. Kwon et al.
the reaction yields ranged from good to excellent (75–95%).
The ketones were obtained with cis-5,6-fused rings (com-
pounds 16) or trans-6,6-fused rings (compounds 17).[15] It is
noteworthy that the decahydronaphthyridinones 18 featured
a cis-fused [4.4.0] bicyclic framework.
cancer cellꢀs metastatic potential is its ability to invade
through extracellular matrix barriers. Therefore, we tested
these compounds for their activities using a cell invasion
assay. Of the three antimigratory compounds, compound
13e displayed the broadest range of subtoxic activity against
cell invasion (Figure 1).
With the collection of 91 compounds in hand, we tested
them in an assay for cancer cell migration. Although cell
movement is a basic biological phenomenon involved in a
range of normal and disease processes, including cancer cell
invasion and metastasis, research probes and chemothera-
peutics that function through known mechanisms that spe-
cifically target cell migration are rare.[16] Despite the rela-
tively small number of compounds in our library, we were
optimistic about the prospect of identifying compounds ex-
hibiting antimigratory activity because of the broad chemi-
cal space covered by the products generated from the
branched pathway (phosphine catalysis/Tebbe/Diels–Alder/
(hydrolysis)). The compound library was screened in a
medium-throughput wound closure assay to identify mole-
cules that inhibited the migration of MDA-MB-231 human
breast cancer cells, essentially as previously described.[17] To
our delight, compounds 9 f, 10e, and 13e exhibited subtoxic
antimigratory activity in the wound closure assay (Table 1).
Figure 1. Inhibition of invasion of MDA-MB-231 cells by 13e. Mean cell
numbers (with standard error of the mean) invading through Matrigel in
a transwell assay are shown (n=6–8 replicates from three independent
experiments). Asterisks denote statistically significant differences from
the control by unpaired, two-tailed Studentꢀs t-tests (with P<0.0006 at
all three concentrations). The minimum lethal concentration (MLC) for
13e under the conditions of the cell invasion assay was 50 mm. In the cell
invasion assay, cells were at a lower density than in the wound closure
assay, and compounds were added at the same time as the cells, rather
than after attachment and growth.
Table 1. Inhibition of migration of MDA-MB-231 human breast cancer
cells.
Compounds 9 f, 10e, and 13e were also evaluated in a tet-
razolium salt-based cell proliferation and viability assay.
They did not inhibit cell growth at concentrations at which
they inhibited cell migration and invasion, but did become
cytotoxic at higher concentrations over a very narrow range,
with minimum lethal concentration (MLC) values similar to
those in Table 1 determined from the trypan blue dye exclu-
sion assay conducted at the end of the wound closure ex-
periments. Therefore, these compounds inhibit cell migra-
tion and invasion at subtoxic concentrations, but, interest-
ingly, they do not exhibit subtoxic antiproliferative (cyto-
static) activity separable from cytotoxicity. Our findings sug-
gest that these compounds have specificity for targets
involved in modulating cell migration and invasion over pro-
liferation. These compounds may, therefore, serve as leads
for the development of targeted inhibitors of cell migration
and invasion to block breast cancer metastasis.
Compound
MIC [mm][a]
IC50 [mm][b]
95% CI [mm][c
MLC [mm][d]
9 f
10e
13e
5
5
5
43.1
14.8
21.6
39.9–46.6
13.1–16.7
19.1–24.5
150
50
100
[a] Minimum inhibitory concentration (MIC) represents the lowest con-
centration tested at which there was statistically significant (by unpaired,
two-tailed Studentꢀs t-tests (P <0.05)) inhibitory activity in the wound
closure assay. [b] Half-maximal inhibitory concentration (IC50) values
were calculated for inhibition of wound closure at 9 h post-wounding
from concentration–response profiles for a range of subtoxic concentra-
tions (n=7–9 wounds from three independent experiments). [c] The 95%
confidence interval (CI) of the IC50. [d] Minimum lethal concentration
(MLC) represents the lowest concentration tested at which there was
statistically significant cell death, measured using the trypan blue dye ex-
clusion assay, by the end of wound closure experiments.
These compounds inhibited cell migration over a range of
concentrations below the threshold at which cell death first
became apparent, suggesting that their antimigratory activi-
ty was separate from—and not just a secondary consequence
of—general cytotoxic effects. Their IC50 values for inhibition
of cell migration were comparable with that of the natural
product migrastatin, which has an IC50 of 29 mm and has
served as a lead for the subsequent development of more
potent analogues.[18]
Conclusion
We have synthesized a library of fused heterocyclic com-
pounds, with 16 distinct scaffolds, through a sequence of
phosphine-catalyzed ring-forming reactions, Tebbe reactions,
Diels–Alder reactions, and, in some cases, hydrolysis. Our
aim for this DOS approach was to efficiently achieve suffi-
ciently high levels of skeletal diversity to explore biological-
ly relevant regions of chemical space. Indeed, our current li-
Cell migration is a necessary, but not sufficient, condition
for the metastasis of solid tumors; a major determinant of a
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Diversity Through a Branched Reaction Pathway
FULL PAPER
Falcon TC companion plates). MDA-MB-231 cells in serum-free DMEM
containing the tested compound or DMSO alone were added to the
upper chambers (100 mLchamberÀ of a 5ꢂ104 cellsmLÀ1 suspension). In
all cases, the final DMSO concentration was 0.05%. Cells were incubated
at 378C under 5% CO2 for 24 h. After removing noninvaded cells from
the upper surface of the membrane with a cotton swab, the invaded cells
on the lower surface were fixed with MeOH, stained with crystal violet
solution, rinsed with water, air-dried, and then counted by using an in-
verted microscope. In parallel experiments, cell viability, determined
using the trypan blue dye exclusion assay at various concentrations of the
tested compounds, was evaluated under the conditions of the cell inva-
sion assay.
brary, despite its small number of components, allowed the
identification of compounds that inhibit cell migration and
cell invasion. We expect that this synthetic pathway can un-
dergo further development and optimization, including the
construction of solid phase libraries of the 16 distinct scaf-
folds for extensive chemical genetic screening. We are opti-
mistic that this approach might lead to the development of
effective tools for chemical biological studies and the iden-
tification of therapeutic lead compounds.
Cell proliferation and cell viability: The assay was conducted according
to the manufacturerꢀs instructions (CCK-8; Dojindo Molecular Technolo-
gies). MDA-MB-231 cells were plated in 96-well tissue culture plates
(125 mLwellÀ1 of a 4ꢂ104 cellsmLÀ1 suspension in growth medium) and
incubated for 24 h at 378C under 5% CO2. The cells were then treated
with the tested compounds at various concentrations or with DMSO
alone. In all cases, the final DMSO concentration was 0.05%. At the
same time, other cells from parallel plates were analyzed using the CCK-
8 assay to establish the cell density at the start of the experiment. After
48 h of incubation, the cell numbers for the experimental samples were
determined and compared with the initial cell numbers. A cytostatic
effect was defined as a reduction in cell number relative to the 48 h con-
trol that did not fall significantly below the mean initial cell number. A
cytotoxic effect was defined as a reduction in cell number that was signif-
icantly below that of the mean initial cell number.
Experimental Section
Synthesis: Detailed synthetic procedures are available in the Supporting
Information.
Statistical analyses: For all assays (wound closure; trypan blue dye exclu-
sion; tetrazolium salt), statistical comparisons between the data obtained
for the control and each treatment condition were performed by using
unpaired, two-tailed Studentꢀs t-tests, with statistically significant differ-
ences defined as P<0.05.
Wound closure: MDA-MB-231 cells (American Type Culture Collection
number: HTB-26) were cultured in 75 cm2 tissue culture flasks with
growth medium (Dulbeccoꢀs minimum essential medium (DMEM) con-
taining 10% fetal bovine serum) at 378C under 5% CO2 in a humidified
tissue culture incubator. The cells were grown to 80% confluence and
then treated with trypsin, collected, and replated in 96-well tissue culture
plates for initial screening. Once confluent, the cell cultures were treated
with compounds at various concentrations or with DMSO carrier solvent
alone with fresh growth medium. In all cases, the final DMSO concentra-
tion was 0.05%. After 30 min, the cell cultures were wounded with a ster-
ile micropipette tip, and the wound closure experiments were performed
essentially as described previously.[17] Compounds were first screened at
50 mm and evaluated for their antimigratory activity or cytotoxicity. This
was accomplished by scoring wounds as opened or closed as a function of
time following wounding to determine the time to closure. Cell viability
was determined by the trypan blue dye exclusion assay conducted at the
end of each experiment, with the ratio of dye-non-excluding (dead) to
dye-excluding (live) cells compared with the control. The following com-
pounds inhibited wound closure or were cytotoxic at 24 h after wound-
ing: 9 f, 10e, 13b, and 13e (none of the other compounds exhibited any
effect on the rate of wound closure, the morphology of the cells or their
viability compared to the control). These four compounds were re-
screened at 10 mm; only compounds 9 f, 10e, and 13e exhibited subtoxic
antimigratory activity at this concentration (they were, however, inactive
when rescreened at 2 mm). Therefore, compounds 9 f, 10e, and 13e were
examined further in quantitative wound closure assays, over a more
finely divided range of concentrations. Cell monolayers in 24-well tissue
culture plates were treated with different concentrations of each com-
pound 30 min before wounding and then wounded, as described above.
Digital images of the wounds were captured immediately after wounding
and at 3, 6, 9, 12, and 24 h post-wounding. The open wound areas, as a
function of time after wounding, were determined with NIH Image J
software (http://rsb.info.nih.gov/nih-image/) to establish mean percent
wound closure over time for each treatment and parallel controls, as pre-
viously described.[17] The mean percent wound closure values at 9 h post-
wounding for all concentrations up to the maximal subtoxic concentra-
tion were subjected to nonlinear regression to calculate IC50 values and
95% confidence intervals for inhibition of wound closure with GraphPad
Prism software.
Acknowledgements
We are grateful to the U.S. National Institutes of Health (O.K.:
R01M071779, P41M081282; G.F.: R01M077622) for financial support.
S.C. thanks the Italian Government (MIUR) for a research grant; S.S.K.
thanks the Nederlandse Organisatie voor Wetenschappelijk Onderzoek
for a TALENT fellowship. We thank Dr. Saeed Khan for performing the
X-ray crystallographic analyses.
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Cell invasion: Compounds 9 f, 10e, and 13e were also analyzed for their
ability to inhibit invasion of cells through Matrigel in a cell invasion
assay. The assay was performed in transwell chambers according to the
manufacturerꢀs instructions (BD Biosciences). The polycarbonate mem-
branes (8 mm pore size) of the upper chambers were coated with Matrigel
(25 mg). Growth medium containing the tested compound or DMSO
alone was added to the lower chambers (600 mLwellÀ1 in 24-well BD
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Received: July 31, 2010
Published online: November 9, 2010
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