Synthesis of an azide-tagged library of 2,3-dihydro-4-quinolones

Article abstract of DOI:10.1021/jo9025447

( Figure Presented ) We describe the assembly of a 960-member library of tricyclic 2,3-dihydro-4-quinolones using a combination of solution-phase high-throughput organic synthesis and parallel chromatographic purification. The library was produced with high efficiency and complete chemo- and diastereoselectivity by diversification of an azide-bearing quinolone via a sequence of [4 + 2] cycloadditions, N-acylations, and reductive aminations. The azide-functionalization of this library is designed to facilitate subsequent preparation of fluorescent or affinity probes, as well as small-molecule/surface conjugation.

Full text of DOI:10.1021/jo9025447

pubs.acs.org/joc
1,4-dihydropyridines,3 dihydropyrimidines,4 pyrrolidines,5
Synthesis of an Azide-Tagged Library of
2,3-Dihydro-4-quinolones
and purines.6 However, despite a number of innovative
synthetic approaches to such small-molecule libraries
reported inthe past, the existingcompound collectionsoccupy
only a small fraction of biogenic chemical space.7 Thus,
synthesis of new heterocyclic libraries to enable discovery
of bioactive chemotypes continues to be of significant
importance. We describe the assembly of a 960-member
library of tricyclic dihydroquinolones, which was produced
with high efficiency and excellent chemical purity by a combi-
nation of miniaturized solution-phase synthesis and high-
throughput chromatographic purification. Azide-functiona-
lization of this library was designed to facilitate subsequent
cellular target identification and construction of small-
molecule microarrays.
Hajoong Lee, Masato Suzuki, Jiayue Cui, and
Sergey A. Kozmin*
Department of Chemistry, University of Chicago,
5735 S. Ellis Ave., Chicago, Illinois 60637
skozmin@uchicago.edu
Received December 1, 2009
We describe the assembly of a 960-member library of
tricyclic 2,3-dihydro-4-quinolones using a combination
of solution-phase high-throughput organic synthesis and
parallel chromatographic purification. The library was
produced with high efficiency and complete chemo- and
diastereoselectivity by diversification of an azide-bearing
quinolone via a sequence of [4 þ 2] cycloadditions,
N-acylations, and reductive aminations. The azide-func-
tionalization of this library is designed to facilitate sub-
sequent preparation of fluorescent or affinity probes, as
well as small-molecule/surface conjugation.
FIGURE 1. Structure of 3,4-dihydro-4-quinolone (1), which is
found in several bioactive chemotypes, including antitumor alka-
loids (2), CRTH2 antagonists (3), and antimitotic agents (4).
Bicyclic 2,3-dihydro-4-quinolone subunit (1, Figure 1) is
found in a number of bioactive small molecules, which
include the acronycine family of alkaloids with potent anti-
cancer properties (2),8 antagonists of CRTH2 receptor (3),9
as well as a class of antimitotic agents (4).10 Such diverse level
of functionalization of 2,3-dihydro-4-quinolone moiety pro-
moted us to examine this structural motif as an attractive
platform for the assembly of the corresponding heterocyclic
library, which was aimed at preserving favorable physico-
chemical properties of the resulting compounds in order to
enable new lead discovery.
Assembly of nitrogen-containing heterocyclic libraries has
been a major focus of high-throughput organic synthesis due
to the prevalence of such chemotypes among bioactive com-
pounds.1 Representative examples include 1,4-benzodiazepines,2
(3) Gordeev, M. F.; Patel, D. V.; Gordon, E. M. J. Org. Chem. 1996, 61,
924–928.
(4) Wipf, P.; Cunningham, A. Tetrahedron Lett. 1995, 36, 7819–7822.
(5) Murphy, M. M.; Schullek, J. R.; Gordon, E. M.; Gallop, M. A. J. Am.
Chem. Soc. 1995, 117, 7029–7030.
(6) Gray, N. S.; Wodicks, L.; Thunnissen, A. H.; Norman, T. C.; Kwon,
S. J.; Espinoza, F. H.; Morgan, D. O.; Barnes, G.; LeClerc, S.; Meijer, L.;
Kim, S. H.; Lockhart, D. J.; Schultz, P. G. Science 1998, 281, 533–538.
(7) Hert, J.; Irwin, J. J.; Laggner, C.; Keiser, M. J.; Shoichet, B. K. Nat.
Chem. Biol. 2009, 5, 479–483.
(8) (a) Huges, G. K.; Lahey, F. N.; Price, J. R.; Webb, L. J. Nature 1948,
162, 223–224. (b) Svoboda, G. H.; Poore, G. A.; Simpson, P. J.; Bodor, G. B.
J. Pharm. Sci. 1966, 55, 758–768.
(9) Liu, J.; Wang, Y.; Sun, Y.; Marshall, D.; Miao, S.; Tonn, G.; Anders,
P.; Tocker, J.; Tang, H. L.; Medina, J. Bioorg. Med. Chem. Lett. 2009, 19,
6840–6844.
(1) (a) Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S. D.;
Keating, T. A. Acc. Chem. Res. 1996, 29, 123–131. (b) Ellman, J. A. Acc.
Chem. Res. 1996, 29, 132–143. (c) Thompson, L. A.; Ellman, J. A. Chem. Rev.
1996, 96, 555–600. (d) Balkenhohl, F.; von dem Bussche-Hunnefeld, C.;
Lansky, A.; Zechel, C. Angew. Chem., Int. Ed. 1996, 35, 2289–2337. (e) Lam,
K. S.; Lebl, M.; Krchnak, V. Chem. Rev. 1997, 97, 411–448. (f) Combinatorial
Chemistry Synthesis and Application; Wilson, S. R., Czarnik A. W., Eds.; John
Wiley & Sons, Inc.: New York, 1997. (g) Gordon, E. M.; Kerwin, J. F., Jr.
Combinatorial Chemistry and Molecular Diversity in Drug Discovery; Wiley-
Liss, Inc.: New York, 1998. (h) Schreiber, S. L. Science 2000, 287, 1964–1969. (i)
Arya, P.; Chou, D. T. H.; Baek, M. G. Angew. Chem., Int. Ed. 2001, 40, 339–346.
(2) (a) Bunin, B. A.; Ellman, J. A. J. Am. Chem. Soc. 1992, 114, 10997–
10998. (b) DeWitt, S. H.; Kiely, J. S.; Stankovic, C. J.; Schroeder, M. C.;
Cody, D. M. R.; Pavia, M. R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6909–
6913.
(10) Zhang, S.-X.; Feng, J.; Kuo, S.-C.; Brossi, A.; Hamel, E.; Tropsha,
A.; Lee, K. H. J. Med. Chem. 2000, 43, 167–176.
1756 J. Org. Chem. 2010, 75, 1756–1759
Published on Web 02/09/2010
DOI: 10.1021/jo9025447
r
2010 American Chemical Society

Lee et al.
JOCNote
SCHEME 1. Synthetic Strategy to Azide-Functionalized
Library of Tricyclic 3,4-Dihydro-4-quinolones
circumvented by using the bulkier reagent NaBH(OEh)3
(Eh = 2-ethylhexanoate),13 which produced the required
1
amine as a single diastereomer detected by 500 MHz H
NMR. Using this protocol, we evaluated the reductive
amination of diketone 7 with 12 commercially available
primary amines (Figure 2B). To facilitate parallel chromato-
graphic purification, all reactions were performed on 2-4
μmol scale to deliver the corresponding products (8-19) in
good chemical yields (61-71%), high diastereomeric ratios
(>95:5), and excellent chemical purities (Figure 2C,D).
The library production began with a selection of enones B
to be employed for the first diversification step of the process
(Figure 3). While a range of unsaturated carbonyl com-
pounds successfully participated in the Beifuss [4 þ 2]
cycloaddition, only 3-substituted enones proved to be viable
for the production of the final library as other substitution
patterns were not tolerated at a later stage of the synthesis.
We selected 10 readily available enones B (Figure 3B), which
were employed to produce a set of corresponding diketones
C following in situ protodesilylation of the TES enol ether
and Alloc deprotection. For the second diversification step,
we selected a set of seven acid chlorides D to be used for
N-acylation of amines C (Figure 3C), as well as the parent
Alloc-protected amine. Each reaction proceeded efficiently
in the presence of Et3N as a base to deliver the required
amides E, setting the stage for the completion of the library
synthesis. The final reductive amination was carried out
using NaBH(OEh)3 as a reducing agent to enable high
diastereoselectivity of this transformation. Each of the 80
diketones E was treated with 12 amines F (Figure 2D) to
produce tricyclic dihydroquinolones G on 4.5 μmol scale,
followed by parallel preparative TLC purification. Initial
analysis revealed that all 960 reactions proceeded success-
fully to deliver final library members in high chemical purity
(see Supporting Information). The purity and efficiency was
further quantified by 500 MHz 1H NMR characterizations
of 120 randomly selected library members. In addition, five
diketones, five amides, and five amines were subjected to full
analytical characterization (Figure 3E). While the efficiency
of the reductive amination ranged from 42% to 75% after
silica gel purification, this protocol provided sufficient
amounts of material (1.0-1.5 mg of final products) for
preparation of DMSO stock solutions to enable 200-500
subsequent high-throughput biological screens. While the
selection of this reaction scale greatly facilitated high-
throughput purification, standard LCMS purification meth-
ods could be readily employed for production of larger
amount of material if desired.
We selected quinolone A (Scheme 1) as the starting point
for introduction of molecular diversity. Indeed, the nitrogen
atom can be readily functionalized by N-acylation. The
vinylogous amide moiety can enable efficient fusion of
additional carbocyclic rings using the [4 þ 2] annulation
developed by Beifuss and co-workers.11 Furthermore, the
aromatic ring can be readily equipped with an azidomethyl
group, which would enable subsequent functionalization of
each library member via alkyne-azide [3 þ 2] cycloaddition.12
The assembly process was designed to begin with a series of
[4 þ 2] cycloadditions of quinolone A with enones B to give
the initial set of tricyclic amines C, which would be subse-
quently diversified via N-acylation using readily available acid
chlorides D to give the resulting diketones E. Chemoselective
and diastereoselective reductive amination of diketones E with
amines F would be employed as the final diversity-generating
step to deliver the target heterocyclic library G. High-
throughput synthesis employing all possible combinations
of 10 enones B, 8 acid chlorides D, and 12 amines F was
expected to deliver 960 tricyclic dihydroquinolones.
Our initial objectives were 2-fold. First, we intended to
validate the generality and efficiency of the synthetic route
shown in Scheme 1. Second, we aimed to develop a simple
protocol that would enable production of all final com-
pounds in high chemical purity. To this end, azido quinolone
5 (Figure 2A), which was readily prepared from 4-nitrobenzyl
alcohol, was first subjected to the [4 þ 2] cycloaddition
with trans-3-nonen-2-one using the protocol developed
by Beifuss and co-workers (TESOTf, 2,6-lutidine, CH2Cl2,
20 °C).11 Protodesilylation of the resulting TES enol ether
with TFA, followed by subsequent Alloc deprotection
(Pd(PPh3)4, morpholine) afforded tricyclic diketone 6, which
was next N-acylated with benzoyl chloride to give amide 7.
The final reductive amination step required differentiation
of the two ketones in 7, as well as a high level of diastereo-
selection. Initially, we examined the use of NaBH(OAc)3,
which promoted reductive amination with high chemo-
selectivity but with only moderate diastereoselectivity
(dr 8:2-9:1), which was not acceptable for a final step of
the library production. Examination of a number of other
reducing agents revealed that this problem could be
Azide-alkyne [3 þ 2] cycloaddition has emerged as a
powerful, general, and bioorthogonal strategy for covalent
molecular conjugation.12 The azide functionalization of
dihydroquinolone library was designed to explore attach-
ment of affinity probes, fluorophores, and radiolabels to
final library members. Alternatively, a similar approach
could be used for surface immobilization of this small-
molecule library.14 Such functionalization would enable
development of new high-throughput screens and facilitate
cellular target identification of active compounds discovered
(13) McGill, J. M.; LaBell, E. S.; Williams, M. Tetrahedron Lett. 1996, 37,
3977–3980.
(14) Uttamchandani, M.; Walsh, D. P.; Yao, S. Q.; Chang, Y. T. Curr.
Opin. Chem. Biol. 2005, 9, 4–13.
(11) Beifuss, U.; Ledderhose, S. Synlett 1995, 938–940.
(12) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128–
1137.
J. Org. Chem. Vol. 75, No. 5, 2010 1757

Lee et al.
JOCNote
FIGURE 2. Validation studies. (A) General reaction sequence. (B) Structures of amines used for reductive amination. (C) Purity analysis of 12
purified products by TLC. (D) 1H NMR spectra, structures and yields for five selected compounds. Yields were determined by 500 MHz 1H
NMR spectroscopy using an internal standard.
FIGURE 3. Assembly of azide-tagged small-molecule library. (A) Reaction sequence. (B) Structures of 10 enones employed for the first
diversification step. (C) Structures of 8 acid chlorides employed for the second diversification step. (D) Structures of 12 amines employed for the
final diversification step. (E) Representative, fully analytically characterized library members.
1758 J. Org. Chem. Vol. 75, No. 5, 2010

Lee et al.
JOCNote
SCHEME 2. Cu-Catalyzed Azide-Alkyne Cycloaddition
C (compound 50, see Supporting Information). 1H NMR
(500 MHz, CDCl3) δ 0.76 (m, 1H), 0.88 (m, 1H), 1.14 (m, 1H),
1.30 (m, 2H), 1.49 (m, 1H), 1.72 (m, 3H), 1.82 (m, 1H), 1.94 (m,
1H), 2.26 (m, 1H), 2.37 (m, 1H), 2.56 (m, 2H), 2.81 (t, 1H, J =
13.4 Hz), 3.44 (br s, 1H), 3.83 (m, 1H), 4.23 (br s, 2H), 4.83 (br s,
1H), 6.68 (d, 1H, J = 8.4 Hz), 7.30 (d, 1H, J = 8.4 Hz), 7.73 (s,
1H); 13C NMR (125 MHz, CDCl3) δ 26.2, 26.4, 26.5, 314, 32.3,
39.0, 41.7, 43.66, 43.74, 46.0, 54.4, 54.9, 116.7, 118.4, 125.1,
127.3, 135.8, 147.9, 194.2, 208.9; MS (APCI) calculated for
C20H24N4O2 352.2, found 387.1 [M þ Cl]-.
A solution of this amine (350 mg, 0.98 mmol) in CH2Cl2
(14 mL) was next divided into 7 equal batches and treated with
pyridine (57 μL, 0.7 mmol) and 7 acid halides (D1-D7)
(0.42 mmol each). The resulting solutions were stirred for 3 h
at room temperature, concentrated in vacuo, and purified by
flash chromatography on silica gel (ethyl acetate/hexane = 1:2)
to give seven corresponding amides E (51-63 mg, 85-99%
yield). The parent N-alloc dihydroquinolone was used as the
eighth amide. Each of the amides E was diluted in CHCl3 to a
final concentration of 0.2 M, divided into 12 equal batches (20 μL,
4 μmol per well), and arrayed into a polypropylene 96-well PCR
plate. The plate was treated with 12 amines F1-F12 (0.74-
1.15 μL) according to the plate map shown in Supporting
Information. Each reaction mixture was treated with the 1.3 M
solution of NaBH(OEh)3 in CH2Cl2 (12.3 μL per well). Upon
completion, the reaction mixtures were transferred onto prepara-
tive TLC plates as circular spots using a multichannel pipettor.
The plates were developed using ether/hexanes = 2:1. The
products were detected under UV light and removed from TLC
plates as silica gel pellets, from which the final compounds were
eluted with 0.8 mL of CH3OH. Following analysis of purity of
each compound G by TLC, the solvent was removed in vacuo.
Twelve randomly selected compounds were dissolved in CD3OD
(0.5 mL) and analyzed by 1H NMR. The amount of material in
each sample was determined by integration using residual CH3OH
as a precalibrated internal standard. This protocol was used next
to prepare all the remaining library members.
in phenotypic assays. Treatment of a representative azide 25
with alkyne 26 in the presence of Cu(MeCN)4PF6 and TBTA
afforded the expected [3 þ 2] cycloadduct 27 in 62% yield
(Scheme 2).15 This experiment validated that the cycloaddition
protocol is efficient and fully compatible with a range of
functional groups present in dihydroquinolones and could
be potentially employed for subsequent derivatization of the
entire chemical library.
In closing, we have assembled a 960-member library of
tricyclic 2,3-dihydro-4-quinolones using a combination of
miniaturized solution-phase high-throughput organic synth-
esis and parallel chromatographic purification. Importantly,
this approach enabled rapid validation of synthetic
sequences and building blocks for library synthesis, which
often represents the bottleneck in the process of generating
new chemical libraries. Second, parallel chromatographic
purification of each individual library member enabled rapid
production of all final compounds in sufficient quantity to
enable hundreds of cell-based or target-based high-through-
put screens. Broad biological evaluation of this library, as
well as the construction of small-molecule microarrays, is
currently in progress. Results of these studies will be reported
in the due course.
Representative Library Member 21. Obtained in 90% yield.
1H NMR (500 MHz, CDCl3) δ 0.73 (m, 1H), 0.86 (m, 1H), 1.13
(d, 1H, J = 12.7 Hz), 1.29 (m, 3H), 1.39 (m, 1H), 1.70 (m, 3H),
1.82 (m, 4H), 1.95 (m, 1H), 2.07 (m, 2H), 3.16 (s, 1H), 3.34 (s,
1H), 3.72 (m, 1H), 3.81 (m, 1H), 3.89 (s, 3H), 3.90 (s, 3H), 4.34
(m, 2H), 5.30 (m, 1H), 6.51 (m, 1H), 6.82 (d, 1H, J = 8.0 Hz),
6.89 (m, 2H), 7.11 (d, 1H, J = 8.4 Hz), 7.16 (d, 1H, J = 3.3 Hz),
7.33 (m, 2H), 7.94 (d, 1H, J = 2.0 Hz); 13C NMR (125 MHz,
CDCl3) δ 26.4, 26.5, 26.8, 29.8, 30.9, 32.7, 38.7, 40.3, 49.7, 51.5,
52.1, 54.1, 56.0, 111.2, 111.6, 112.2, 118.3, 120.3, 124.3, 125.0,
126.6, 131.6, 133.3, 141.4, 144.8, 147.9, 148.2, 149.1, 160.0,
195.6; HRMS (ESI) calculated for C34H40N5O5 [M þ H]þ
598.3029, found 598.3029.
Experimental Section
General Protocol for Library Synthesis. The following proce-
dure represents the synthesis of a representative set of
96 compounds (plate 4). To a solution of quinolone 5 (1.0 g,
3.52 mmol) in CH2Cl2 (30 mL) were added 2,6-lutidine (1.4 mL,
12.3 mmol), TESOTf (2 mL, 8.8 mmol), and a solution of enone
B4 (1.1 g, 7.04 mmol) in CH2Cl2 (10 mL). The resulting solution
was stirred for 2 h at room temperature and was treated with
TFA (0.54 mL, 7.04 mmol). The reaction was quenched by
addition of saturated aqueous solution of NaHCO3 (15 mL) and
extracted with CH2Cl2 (3 ꢀ 30 mL). The organic layer was dried
with anhydrous MgSO4, concentrated in vacuo, and purified by
flash chromatography on silica gel (ethyl acetate/hexane = 1:3)
to give 1.1 g (70% yield) of the corresponding tricyclic ketone.
This product (0.72 g, 1.83 mmol) was dissolved in THF (20 mL)
and treated with Pd(PPh3)4 (10.5 mg, 0.09 mmol) and morpho-
line (0.2 mL, 3.66 mmol). The resulting solution was stirred for
30 min at 20 °C, concentrated in vacuo, and purified by flash
chromatography on silica gel (ethyl acetate/hexane = 1:1) to
give 580 mg (90% yield) of the corresponding tricyclic amine
Acknowledgment. Partial support of this research was
provided by NIH P50 GM086145. S.A.K thanks the Alfred
P. Sloan Foundation, the Dreyfus Foundation, Amgen and
GlaxoSmithKline for additional financial support.
Supporting Information Available: Additional experimental
protocols, plate maps, images of TLC plates and copies of NMR
spectra. This information is available free of charge via the
Internet at http://pubs.acs.org.
(15) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
2004, 6, 2853–2856.
J. Org. Chem. Vol. 75, No. 5, 2010 1759