Solid-phase synthesis of azidomethylene inhibitors targeting cysteine proteases

Article abstract of DOI:10.1021/ol800575z

An efficient strategy for the solid-phase synthesis of azidomethylene inhibitors targeting cysteine proteases is described. The method is highlighted by its compatibility with readily available building blocks, as well as its ability to accommodate differ

Full text of DOI:10.1021/ol800575z

ORGANIC
LETTERS
2008
Vol. 10, No. 10
1881-1884
Solid-Phase Synthesis of
Azidomethylene Inhibitors Targeting
Cysteine Proteases
Peng-Yu Yang, Hao Wu, Mei Yin Lee, Ashley Xu, Rajavel Srinivasan, and
Shao Q. Yao*
Departments of Chemistry and Biological Sciences, NUS MedChem Program of the
Office of Life Sciences, National UniVersity of Singapore, Singapore 117543
chmyaosq@nus.edu.sg
Received December 18, 2007
ABSTRACT
An efficient strategy for the solid-phase synthesis of azidomethylene inhibitors targeting cysteine proteases is described. The method is
highlighted by its compatibility with readily available building blocks, as well as its ability to accommodate different functional groups. A
249-member library has thus far been successfully synthesized, characterized, and screened against Caspase-1, -3 and -7.
Recent advances in genomics and proteomics necesssiates
the development of highly efficient chemical reactions
capable of rapid synthesis of potential drug candidates.1 Such
reactions are exemplified by their high efficiency (e.g.,
quantitative product formation), good tolerance toward a
variety of functional groups, and compatibility with screening
(e.g., reaction is carried out in water; no product purification
is necessary before biological assay). Thus far, the Cu(I)-
catalyzed 1,3-dipolar cycloaddition reaction, or better known
as “click chemistry”, between an azide and an alkyne, has
been extensively studied,2 and proven to be highly successful,
especially for high-throughput synthesis and discovery of
enzyme-inhibitors.2b The amide-forming reaction between
an amine and an acid is one of the most efficient reactions
known, and has recently been applied successfully in the
rapid discovery of inhibitors against a number of enzymes,
such as HIV proteases,3a,b ꢀ-aryl sulfotransferase,3c
R-fucosidases,3d,e and SARS-3CL protease.3f This chemistry
has some key characteristics of a high-throughput amenable
reaction, in that quantitative product formation can be
achieved using powerful acylating/coupling reagents. As
such, in situ biological screening may be carried out directly
without product purification. The method, however, is
severely limited by the presence of coupling reagents and,
more often than not, byproduct and excessive starting
materials in the reaction, and subsequently during in situ
(3) (a) Brik, A.; Lin, Y.-C.; Elder, J.; Wong, C.-H. Chem. Biol. 2002,
9, 891. (b) Lee, S.-G.; Chmielewski, J. Chem. Biol. 2006, 13, 421. (c) Best,
M. D.; Brik, A.; Chapman, E.; Lee, L. V.; Wong, C.-H. ChemBioChem
2004, 5, 811. (d) Wu, C.-Y.; Chang, C.-F.; Chen, J. S.-Y.; Lee, S.-T.; Wong,
C.-H.; Lin, C.-H. Angew. Chem., Int. Ed. 2003, 42, 4661. (e) Chang, C.-F.;
Ho, C.-W.; Wu, C.-Y.; Chao, T.-A.; Wong, C.-H.; Lin, C.-H. Chem. Biol.
2004, 11, 1301. (f) Wu, C.-Y.; King, K.-Y.; Kuo, C.-J.; Fang, J.-M.; Wu,
Y.-T.; Ho, M.-Y.; Liao, C.-L.; Shie, J.-J.; Liang, P.-H.; Wong, C.-H. Chem.
(1) Sun, H.; Chattopadhaya, S.; Wang, J.; Yao, S. Q. Anal. Bioanal.
Chem. 2006, 386, 416.
(2) For recent reviews, see: Kolb, H. C.; Sharpless, K. B. Drug DiscoVery
Today 2003, 8, 1128. (b) Angell, Y. L.; Burgess, K. Chem. Soc. ReV. 2007,
1674.
Biol. 2006, 13, 261
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10.1021/ol800575z CCC: $40.75
Published on Web 04/12/2008
2008 American Chemical Society

so far, the hallmark of this family of enzymes is their absolute
requirement for an aspartic acid residue at the P1 site of their
substrates. Over the past few years, a variety of caspase
inhibitors, both reversible and irreversible, have been de-
veloped.6 Most inhibitors contain an electrophilic “warhead”
such as an aldehyde, ketone, halomethyl ketone, epoxide,
or vinyl sulfone (Figure 1b, I), which reacts with the cysteine
residue located in the active site of a caspase. To improve
“druglike” properties, the P2 and P3 positions in a peptide-
based caspase inhibitor may be replaced with suitable
nonpeptide linkers (see II7 in Figure 1b) with minimal effect
on the inhibitor potency. The P4 position in a caspase
inhibitor is known to be the major determinant of both its
binding and specificity toward different caspases.5 For
example, it has been shown that caspases 3 and 7 prefer
charged/hydrophilic groups at this position, while caspase 1
prefers predominantly hydrophobic groups. Consequently,
potent and (in some cases) specific inhibitors, such as II and
III, were successfully developed.7,8 We were particularly
intrigued by inhibitors such as III, which were recently
reported by Fairlie and co-workers,8a as they contain a
relatively unreactive azidomethylene group as the electro-
philic warhead. We thought since an azidomethylene group
is chemically and metabolically inert, molecules possessing
this “warhead” might offer special advantages as potential
cysteine protease inhibitors.1 We therefore decided to develop
an efficient route capable of making azidomethylene inhibi-
tors targeting different classes of cysteine proteases including
caspases (Scheme 1).9 It is noted that, while our manuscript
was in preparation, Fairlie et al. reported that peptidic
azidomethylene inhibitors were unexpectedly susceptible to
photolytic degradation, giving rise to traces of aldehyde and
monoacyl aminal products.8b
Figure 1. (a) Two strategies using amide-forming reactions; (b)
structures of different known caspase inhibitors.
screening, which may lead to false results (Figure 1a;
pathway A).4 In fact, in a recent study by Wong et al., the
authors unexpectedly discovered that it was the intermediate
active ester formed between a carboxylic acid (a starting
material) and HBTU (a coupling reagent) which gave rise
to potent inhibition against the target, SARS-3CL protease.3f
Herein, we report a traceless solid-phase method for rapid
synthesis of novel small molecule inhibitors using amide
bond-forming reactions (Figure 1a; pathway B); the strategy
takes advantage of the 4-formyl-3-methoxyphenoxy (FMP)
resin for (1) amine capturing using an aldehyde handle; (2)
subsequent solid-phase amide-forming reaction with a linker
and an incoming acid using suitable coupling reagents; and
(3) cleavage/release of desired products which are of
sufficient purity for direct in situ screening. The method is
“traceless”, allowing the use of the exact same sets of starting
materials as in pathway A (e.g., same amine/acid building
blocks) in solid phase without any modification. We have
successfully implemented this strategy for the facile synthesis
of 249-member azidomethylene inhibitors targeting cysteine
proteases.
A 249-member small molecule library was constructed
following Scheme 1, using the IRORI directed sorting
technology.10 As shown in Figure 1b (boxed), most members
consist of an aspartic acid-derived azidomethylene warhead,
a suitable linker located at the P2 and P3 positions to render
the inhibitors more druglike, and a diverse P4 group
introduced from commercially available building blocks
(acids, sulfonyl chlorides, chloroformates, and isocyanates).
To make the central amino azide scaffold, 4, an Fmoc-
protected amino acid 1 was converted into the corresponding
alcohol 2, then subsequently into the corresponding azide 3
by the Mitsunobu reaction with hydrogen azide.11 Following
(6) (a) Otto, H.-H.; Schirmeister, T. Chem. ReV. 1997, 97, 133. (b)
Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. ReV. 2002,
102, 4639.
(7) (a) Choong, I. C.; Lew, W.; Lee, D.; Pham, P.; Burdett, M. T.; Lam,
J. W.; Wiesmann, C.; Luong, T. N.; Fahr, B.; DeLano, W. L.; McDowell,
R. S.; Allen, D. A.; Erlanson, D. A.; Gordon, E. M.; O’Brien, T. J. Med.
Chem. 2002, 45, 5005. (b) Erlanson, D. A.; Lam, J. W.; Wiesmann, C.;
Luong, T. N.; Simmons, R. L.; DeLano, W. L.; Choong, I. C.; Burdett,
M. T.; Flanagan, W. M.; Lee, D.; Gordon, E. M.; O’Brien, T. Nat.
Biotechnol. 2003, 21, 308.
Among the numerous classes of cysteine proteases in-
volved in human diseases, caspases are well-known to play
key roles in the regulation of apoptosis and inflammatory
responses.5 With more than 15 different members identified
(8) (a) Le, G. T.; Abbenante, G.; Madala, P. K.; Hoang, H. N.; Fairlie,
D. P. J. Am. Chem. Soc. 2006, 128, 12396. (b) Abbenante, G.; Le, G. T.;
Fairlie, D. P. Chem. Commun. 2007, 4501
.
(9) Existing strategy for the azidomethylene inhibitors made use of
solution-phase chemistry which is low throughout and extremely tedious,
therefore not HT amenable. See ref 8 for details.
(4) Brik, A.; Wu, C.-Y.; Wong, C.-H. Org. Biomol. Chem. 2006, 4, 1446.
(5) Denault, J. B.; Salvesen, G. S. Chem. ReV. 2002, 102, 4489.
(10) Xiao, Y.; Li, R.; Zhuang, H.; Ewing, B.; Karunaratne, K.; Lillig,
J.; Brown, R.; Nicolaou, K. C. Biotechnol. Bioeng. 2000, 71, 44.
1882
Org. Lett., Vol. 10, No. 10, 2008

Scheme 1. Traceless Solid-Phase Synthesis of Azidomethylene Inhibitor
removal of Fmoc group with 20% piperidine in DMF, the
desired amino azides, 4a-d, were obtained in good yields.
Next, 4 was attached to the FMP resin by reductive amination
(6 equiv of NaBH(OAc)3 in DCE with 1% AcOH) via its
amino group, which was concurrently converted to the resin-
bound, secondary amine 5. Subsequently, acylation of the
amines was efficiently carried out with the corresponding
Fmoc-protected carboxylic acid chloride linker (6a-c, 5
equiv) with DIEA (10 equiv) in anhydrous CH2Cl2, giving
7. Upon Fmoc removal (20% piperidine in DMF), the
resulting resin was treated with a variety of commercially
available building blocks (see Supporting Information). In
general, acylation with acids (aromatic/aliphatic) was found
to proceed cleanly with HBTU/HOBt, but for less reactive
Figure 2. Representative structures of the 249-member azidomethylene library.
Org. Lett., Vol. 10, No. 10, 2008
1883

carboxylic acids, PyBOP/HOAt was shown to work better.
Sulfonylation was found to proceed cleanly in DCM/DIEA
with a catalytic amount of DMAP. Reactions with isocyan-
ates proceeded smoothly in the presence of DIEA in DMF.
Lastly, the cleavage was accomplished with a TFA/DCM/
H2O (5:4:1) mixture, affording the desired products as the
corresponding amides, carbamates, sulfonamides, and ureas
in good yields (based on a theoretical loading of 0.9 mmol/
g). A variety of functional groups such as esters, nitriles,
halogens, hydroxyl, nitro, and even salicylic acid, as shown
in Figure 2, were easily tolerated.
To ensure crude products generated from our synthetic
route were sufficiently pure for direct in situ screening, we
characterized them by LCMS and NMR (Supporting Infor-
mation); in most cases the desired produts were obtained
with >90% purity and shown to be spectroscopically
homogeneous (by NMR). It is also worth noting that, unlike
what Fairlie et al. reported,8b we have NOT noticed any
product degradation from selected compounds in our library
even after 10-month storage (see Supporting Information).
We next screened the compounds for inhibiton against
Caspase-1, -3, and -7 (Supporting Information); at 10 µM
inhibitor concentration, while most compounds showed little
or no inhibiton against Caspase-3 and -7, a few of them
inhibited Caspase-1 fairely well (i.e., W2-E1, W2-E2,
W2-F6, W3-A2). Therefore these compounds were further
screened against Caspase-1 to obain their IC50 and Ki values.
We were pleased to find that the most potent inhibitor, W2-
E1, was able to inhibit Caspase-1 with IC50 and Ki values of
1.78 and 1.16 µM, respectively (Figure 3). The same
compound showed much weaker inhibiton against Caspase-3
and -7 (IC50 > 200 µM and Ki values could not be accurately
determined due to compound precipitation at higher con-
centrations). These values are moderate when compared to
other potent Caspase-1 inhibitors.6 It nevertheless indicates
that methylene azides are indeed useful pharmacophores for
future development of cysteine protease inhibitors.
Figure 3. (a) Structure of W2-E1; (b and c) IC50 and Ki of W2-E1
against Caspase-1, respectively. In panel c, the inhibitor concentra-
tions are (×) 1250, (/) 500, (9) 100, and ()) 0 nM.
functionalities in each of the components. Products generated
were of uniformly high quality, enabling future direct in situ
screening with a suitable biological target. Preliminary results
indicate selected compounds are indeed moderate caspase
inhibitors. This method should serve as a useful model for
future development of other high-throughput amenable
chemistry in the emerging field of catalomics.1,12
Acknowledgment. Funding support was provided by the
National University of Singapore (NUS) and the Agency for
Science, Technology and Research (A*STAR) of Singapore.
Supporting Information Available: Full characterizations
of compounds and experimental details. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL800575Z
In summary, we have developed an efficient and traceless
solid-phase synthesis of azidomethylene derivatives as
potential cysteine protease inhibitors. The strategy allows
facile assembly of azidomethylene warheads with suitable
nonpeptide linkers as well as a variety of readily available
building blocks, all without the need of introducing extra
(11) Boeijen, A.; van Ameijde, J.; Liskamp, R. M. J. J. Org. Chem.
2001, 66, 8454
.
(12) (a) Uttamchandani, M.; Lee, W. L.; Wang, J.; Yao, S. Q. J. Am.
Chem. Soc. 2007, 129, 13110. (b) Uttamchandani, M.; Wang, J.; Li, J.;
Hu, M.; Sun, H.; Chen, K. Y.-T.; Liu, K.; Yao, S. Q. J. Am. Chem. Soc.
2007, 129, 7848. (c) Kalesh, K. A.; Yang, P.-Y.; Srinivasan, R.; Yao, S. Q.
QSAR Comb. Sci. 2007, 26, 1135
.
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Org. Lett., Vol. 10, No. 10, 2008