Skeletal diversity via a branched pathway: Efficient synthesis of 29 400 discrete, polycyclic compounds and their arraying into stock solutions

Article abstract of DOI:10.1021/ja028086e

We report the synthesis and arraying of 29400 structurally diverse and complex polycyclic carbocycles using diversity-oriented synthesis (DOS) and the "one bead-one stock solution" technology platform. Skeletal diversity, a difficult challenge in DOS, was

Full text of DOI:10.1021/ja028086e

Published on Web 10/18/2002
Skeletal Diversity via a Branched Pathway: Efficient Synthesis of 29 400
Discrete, Polycyclic Compounds and Their Arraying into Stock Solutions
Ohyun Kwon,† Seung Bum Park, and Stuart L. Schreiber*
Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, HarVard Institute of Chemistry
and Cell Biology, HarVard UniVersity, 12 Oxford Street, Cambridge, Massachusetts 02138
Received August 9, 2002
Diversity-oriented synthesis (DOS) aims to synthesize efficiently
complex, small molecules broadly distributed in multidimensional
descriptor space.1 Such collections2 are key to chemical genetics,
where small molecules are used to explore biology and medicine
systematically.3 Skeletal diversity in DOS has proven to be
especially challenging. Here, we report a branching DOS pathway
that yields 29 400 discrete compounds comprising 10 distinct
polycyclic skeletons.4
The six-step, stereoselective synthesis, which affords products
having a central skeleton with between two and four rings and up
to six stereocenters, has been achieved using an inexpensive and
accessible, “one bead-one stock solution” technology platform.5 The
pathway builds on the report by Fallis and co-workers on the use
of consecutive Diels-Alder reactions.6 We have adapted their
reported triene synthesis and subsequent complexity-generating
reactions to phenolic aldehyde-loaded macrobeads and discovered
a set of dienophiles that react only once with the Fallis-type trienes.
The latter observation provides a branch point to the pathway, where
diene products are formed from a single Diels-Alder cycloaddition,
and monoene products are formed from consecutive Diels-Alder
reactions involving either the same or different dienophiles (Figure
1). An important feature of the branched pathway is that the
diastereoselection observed in the original report has been extended
to reaction sequences involving different dienophiles.
Figure 1. Hydroxyl-substituted aromatic aldehydes (most frequently,
phenolic aldehydes; vanillin is illustrated) were loaded onto high capacity
macrobeads (denoted by the asterisk-within-a-circle symbol), converted to
trienes, and reacted with dienophiles. The degree of substitution on the
dienophiles determines whether they participate in the second cycloaddition
(see text for details). The diamond inserted in the arrow denotes a split-
and-pool step.
crystallography in five cases, verified that the selectivity reported
by Fallis and co-workers was general.12
To optimize the yield and purity of the library members, potential
building blocks for the library were tested individually as follows.
In separate reaction vessels, 64 hydroxyaldehydes were loaded onto
macrobeads through silylation of their hydroxyl groups with the
previously described macrobead-alkylsilyl triflate (illustrated with
the silylation-loading of vanillin 1 in Figure 1).7 Each macrobead-
loaded aldehyde was separately reacted with indium dust8 and
5-bromo-1,3-pentadiene9 in DMF, which provided the γ-addition
product (2 in the illustrated case with vanillin).10 Mesylation
followed by elimination using DBU furnished the cross-conjugated
triene (cf., 3).10 After cleavage with HF-py and analysis of the purity
of the triene products by 1H NMR, 40 of the original 64
hydroxyaldehydes (Figure 2 top) were found to yield a single
identifiable compound.10 These 40 aldehydes were used in the DOS
pathway described below.
Macrobead-loaded triene 3 (Figure 1) was used to assess the
reactivity and stereoselectivity of 53 disubstituted- and 44 tri- or
tetrasubstituted cyclic dienophiles. In earlier pathway-development
studies, we had ascertained that noncyclic dienophiles11 afforded
stereoisomeric mixtures of double cycloadducts, whereas cyclic
dienophiles yielded products stereoselectively.10 Spectroscopic
analyses of single and double cycloadducts, including X-ray
An important pattern of reactivity was uncovered using 3:
disubstituted dienophiles underwent double cycloaddition (cf., 4),
whereas tri- or tetrasubstituted dienophiles underwent mono cy-
cloaddition (cf., 5). Using the criterion of single isomer formation
(cf., 4) in high purity from triene 3, we selected 41 (of 53)
disubstituted dienophiles (Figure 2, middle) for use in the DOS
pathway.10 Representative members of mono-cycloadduct dienes
(cf., 5) were found to undergo stereoselective Diels-Alder reactions
with a second dienophile to yield tetracycles derived from two
different dienophiles (cf., 6). Using the criteria of efficient, single
isomer production of both single and double cycloadducts (cf., 5
and 6), we selected 22 (of 44) tri- or tetrasubstituted dienophiles
(Figure 2, bottom) for use in the DOS pathway.10 These dienophiles,
which “interrupt” the double Diels-Alder process, provide a key
skeleton-diversifying branch in the DOS pathway. Combinations
of the selected skeletal building blocks are calculated to produce a
maximum of 29 400 distinct compounds.13
Approximately 88 200 macrobeads15 were divided into 40 equal
portions and loaded with the 40 aldehydes described above in
separate reaction vessels. The individual vessels of aldehyde-loaded
macrobeads (cf., 1) were tagged with diazo-based electrophoretic
reporters using a binary code,16 pooled, and converted to triene-
loaded macrobeads as described above (cf., 3). The tagged and
pooled triene-containing macrobeads were divided into 23 portions.
* To whom correspondence should be addressed. E-mail: sls@slsiris.harvard.edu.
† Current address: University of California, Los Angeles.
9
13402
J. AM. CHEM. SOC. 2002, 124, 13402-13404
10.1021/ja028086e CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. Forty hydroxyaldehyde- (top), 41 disubstituted dienophile-
(middle), and 22 tri- or tetrasubstituted dienophile- (bottom) building blocks
used in the branched DOS pathway.
One portion was recombined later with the dienes prepared below
for the consecutive Diels-Alder cycloaddition (cf., 3 to 4). Twenty-
two portions were reacted individually, using optimized conditions,
with 22 dienophiles (Figure 2, bottom). Each segregated collection
of macrobeads was tagged using additional reporters. The 22 vessels
containing single cycloadducts (cf., 5) were pooled and subsequently
divided into 4 × 41 portions (the 41 portions were grouped into
four sets because we found that the subsequent Diels-Alder
reactions fell into four different optimal reaction conditions
depending on the reactivity of the second dienophile10). A 42nd
portion was set aside to be combined with the collection of
tetracyclic compounds, thus ensuring the presence of bicyclic dienes
(cf., 5) in the final collection of products. The 4 × 41 vessels were
treated individually with the 41 dienophiles (using four different
conditions) that undergo the second cycloaddition (Figure 2, middle)
and tagged using additional reporters. The pooled, 88 200 encoded
macrobeads serve to segregate a high percentage of the theoretical
29 400 compounds prior to automated preparation of stock solutions.
Our quality control efforts during the pathway development phase
of this research identified the reaction partners expected to undergo
efficient and predictable outcomes, but they also revealed reactivity
patterns that further diversified the skeletons4 of the products of
this DOS pathway (Figures 3 and 4). Whereas macrobead-bound
trienes (cf., 3) reacted with tri- and tetrasubstituted dienophiles to
yield the expected bicycles of structural types S1 and S2 (verified
in 10 and 8), they reacted with halogenated dienophiles to yield
structural types S3, S9, and S10. These latter compounds result
from cycloadditions followed by dehydrohalogenation: S3 (verified
in 11) by dehydroiodination10 and S9-10 (verified in 13) by
dehydrobromination (dehydrohalogenation was facilitated with
strontium carbonate17 when maleimides were used as the second
dienophile). Macrobead-bound dienes of S1 react with maleimides
to yield the expected tetracycle of S4 (verified in 7′), but they react
with 4-phenyl-1,2,4-triazoline-3,5-dione (and presumably related
dienophiles) to yield products having anti,anti- and syn,anti-trans-
fused C-D ring junctions as in S5 and S10 (verified in 12 and
13). Extending these observations to the possible combinations of
Figure 3. Products derived from the intermediates in Figure 1 and several
related products characterized by X-ray crystallography. Ring B on each
skeleton is highlighted in black in the Chem 3D images derived from X-ray
coordinates.14
Figure 4. The branched DOS pathway leads to compounds having 10
distinct skeletons.4
dienophile building blocks suggests that at least 10 different
skeletons4 will be represented among the 29 400 anticipated
products.
Our first step in analyzing purity and identity of these products
entailed the random selection of 50 macrobeads from the final pool.
9
J. AM. CHEM. SOC. VOL. 124, NO. 45, 2002 13403

C O M M U N I C A T I O N S
Products were eluted from the macrobeads with HF-py (and then
TMSOEt), diluted to 10 mM stock solutions (DMF), and analyzed
by LC/MS and stock solution decoding.18 These data revealed
acceptable levels of purity and structures consistent with expecta-
tions. Our second step in postsynthesis quality control was
performed following both full arraying of all macrobeads and
automated stock solution preparation.
the National Cancer Institute, Merck KGaA, Merck & Co., and
the Keck Foundation for support of the Harvard Institute of
Chemistry and Cell Biology, and the National Cancer Institute for
support of the Molecular Target Laboratory. O.K. was and S.B.P.
is a Research Associate, and S.L.S. is an Investigator at the Howard
Hughes Medical Institute in the Department of Chemistry and
Chemical Biology, Harvard University.
The 88 200 individual macrobeads were first arrayed into 384-
well microtiter plates using a vacuum-based bead arrayer to entrain
352 beads in an equal number of wells (two columns of wells from
each plate were left empty to accommodate controls used in
subsequent assays).5a Microtiter plates containing one bead per well
were then subjected to a robotic cleavage process, in which each
well was treated with 20 µL of HF-py cocktail (5% HF-py, 5% py
in THF) delivered using a ceramic pump. After 300 min at room
temperature, each cleavage reaction was quenched with 20 µL of
TMSOEt19 for 30 min, evaporated, and eluted from beads with three
30 µL DMF washes. DMF eluates were pooled into fresh 384-
well “mother plates”, each of which was mapped into five “daughter
plates” by volumetric transfer using a Hydra384 syringe-array robot
(50% of stock solution for cell-based assays, 20% for small
molecule microarrays 2 × 10% for compound archiving, and 10%
for chemical analysis).5b
Currently, 150 microtiter plates (52 800 single compound-
containing stock solutions, approximately two theoretical copies)
have been arrayed, and 61 microtiter plates (21 472 compounds,
73% of a theoretical copy) have been formatted into “daughter
plates”. For post-automated formatting, quality control (QC)
analysis, we again used LC/MS and stock solution decoding.18b The
structures of 88 out of 100 samples were inferred successfully by
LC/MS and GC decoding. The structures of the remaining 12 were
inferred by GC decoding, but could not be confirmed by LC/MS.
Preliminary analysis of the purity of resulting stock solutions
and their performance in both protein-binding and phenotypic assays
has revealed that the overall process is sufficient for identifying
novel small molecules having specific and potent protein-binding
and cellular activities. We expect that the pathway should be subject
to further development and optimization, including modified
pathways guided by analyses of the molecular descriptors of the
small molecules in advance of their synthesis. We are optimistic
that this pathway will provide many effective probes for chemical
genetic studies aimed at dissecting biology.
Supporting Information Available: Representative experimental
procedures and characterization data (PDF). An X-ray crystallographic
file (CIF). This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(1) Schreiber, S. L. Science 2000, 287, 1964-1969.
(2) Dolle, R. E. J. Comb. Chem. 2001, 3, 477-517.
(3) (a) Schreiber, S. L. Bioorg. Med. Chem. 1998, 6, 1127-1152. (b)
Mitchison, T. J. Chem. Biol. 1994, 1, 3-6.
(4) In this paper, we define compounds with different: (1) numbers or sizes
of rings, (2) ring fusion stereochemistry, or (3) degree of ring fusion
saturation as having “different skeletons”.
(5) (a) Blackwell, H. E.; Perez, L.; Stavenger, R. A.; Tallarico, J. A.; Eatough,
E. C.; Foley, M. A.; Schreiber, S. L. Chem. Biol. 2001, 8, 1167-1182.
(b) Clemons, P. A.; Koehler, A. N.; Wagner, B. K.; Sprigings, T. G.;
Spring, D. R.; King, R. W.; Schreiber, S. L.; Foley, M. A. Chem. Biol.
2001, 8, 1183-1195.
(6) Woo, S.; Squires, N.; Fallis, A. G. Org. Lett. 1999, 1, 573-575.
(7) Tallarico, J. A.; Depew, K. D.; Pelish, H. E.; Westwood, N. J.; Lindsley,
C. W.; Shair, M. D.; Schreiber, S. L.; Foley, M. A. J. Comb. Chem. 2001,
3, 312-318.
(8) For indium-mediated allylation of resin-bound aldehydes with sonica-
tion: Cavallaro, C. L.; Herpin, T.; MuGuinness, B. F.; Shimshock, Y.
C.; Dolle, R. E. Tetrahedron Lett. 1999, 40, 2711-2714.
(9) Prevost, C.; Miginiac, P.; Miginiac-Groizeleau, L. Bull. Soc. Chim. Fr.
1964, 2485-2492.
(10) See Supporting Information for details.
(11) Acyclic dienophiles tested: trans-â-nitrostyrene, dimethyl maleate, and
dimethyl fumarate.
(12) HF-py-mediated cleavage of macrobead-loaded 4 resulting from 100 mg
of 3-[diisopropyl(p-methoxyphenyl)silyl]propyl functionalized macrobeads
yielded 32 mg (0.71 mmol/g of beads, 109 nmol/bead) of the tetracyclic
product 7 (Figure 3) (single diastereomer and 95% pure by 1H NMR).
(13) 800 dienes (40 aldehydes × 20 dienophiles), 2640 tetracycles from
interrupted D-A with 1,2,4-triazoline-3,5-diones (40 aldehydes × 22
dienophiles × 3 disubstituted dienophiles), 24 320 tetracycles from
interrupted D-A with maleimides (40 aldehydes × 16 dienophiles × 38
disubstituted dienophiles), and 1640 tetracycles from consecutive D-A
(40 aldehydes × 41 disubstituted dienophiles).
(14) Compounds 7′, 12, and 13 were produced through solution-phase synthesis
during the pathway development phase of this research.10
(15) Burgess, K.; Liaw, A. I.; Wang, N. J. Med. Chem. 1994, 37, 2985-2987.
(16) (a) Ohlmeyer, M. J. H.; Swanson, R. N.; Dillard, L. W.; Reader, J. C.;
Asouline, G.; Kobayashi, R.; Wigler, M.; Still, W. C. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 10922-10926. (b) Nestler, H. P.; Bartlett, P. A.;
Still, W. C. J. Org. Chem. 1994, 59, 4723-4724.
(17) Pearlman, B. A.; McNamara, J. M.; Hasan, I.; Hatakeyama, S.; Sekizaki,
H.; Kishi, Y. J. Am. Chem. Soc. 1981, 103, 4248-4251.
(18) (a) Stavenger, R. A.; Schreiber, S. L. Angew. Chem., Int. Ed. 2001, 40,
3417-3421. (b) Blackwell, H. E.; Perez, L.; Schreiber, S. L. Angew.
Chem., Int. Ed. 2001, 40, 3421-3425.
(19) TMSOEt was used as the quenching reagent instead of previously reported
TMSOMe to minimize cross-contamination in the 386-well microtiter
plate.
Acknowledgment. We thank Richard Staples for performing
X-ray crystallographic analysis and Cullen M. Taniguchi for
assistance in the early phase of these studies. We are especially
grateful to John Tallarico, Paul Clemons, and Max Narovlyansky
of the Harvard ICCB for their assistance. We thank the National
Institute for General Medical Sciences for support of this research,
JA028086E
9
13404 J. AM. CHEM. SOC. VOL. 124, NO. 45, 2002