Efficient and facile synthesis of acrylamide libraries for protein-guided tethering

Article abstract of DOI:10.1021/ol503486t

A kinetic template-guided tethering (KTGT) strategy has been developed for the site-directed discovery of fragments that bind to defined protein surfaces, where acrylamide-modified fragments can be irreversibly captured in a protein-templated conjugate ad

Full text of DOI:10.1021/ol503486t

Letter
pubs.acs.org/OrgLett
Efficient and Facile Synthesis of Acrylamide Libraries for Protein-
Guided Tethering
Charlotte E. Allen,† Peter R. Curran,† Andrew S. Brearley,† Valerie Boissel,† Lilya Sviridenko,†
Neil J. Press,† Jeffrey P. Stonehouse,*,† and Alan Armstrong‡
†Global Discovery Chemistry, Novartis Horsham Research Centre, Wimblehurst Road, Horsham RH12 5AB, U.K.
‡Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K.
S
* Supporting Information
ABSTRACT: A kinetic template-guided tethering (KTGT)
strategy has been developed for the site-directed discovery of
fragments that bind to defined protein surfaces, where
acrylamide-modified fragments can be irreversibly captured
in a protein-templated conjugate addition reaction. Herein, an
efficient and facile method is reported for the preparation of
acrylamide libraries from a diverse range of amine fragments using a solid-supported quaternary amine base.
methods for acrylamide synthesis are well documented but
ver the past decade, tethering strategies have been
Osuccessfully developed for the site-directed discovery of are often low yielding and unsuitable for high-throughput
fragments that bind to defined protein surfaces.1−4 These
library synthesis. There have been previous attempts to
include kinetic template-guided tethering (KTGT),1 where
overcome the current limitations, in particular through the
use of solid-phase syntheses,6,7 but these often require a large
upon fragment binding a Cys-residue adjacent to the protein
number of steps for a simple chemical transformation.
Moreover, the commercial availability of chemically diverse,
acrylamide-functionalized fragments is relatively low compared
to other structural classes such as amines or carboxylic acids.
Herein, we describe the design of an acrylamide library for use
in KTGT, and the optimization of an efficient and facile
method for the preparation of acrylamide libraries from a
diverse range of amine fragments, suitable for fragment based
drug discovery(FBDD), using a solid-supported quaternary
amine base.
A library of 192 fragment amines was designed for
modification to the corresponding acrylamide for use in a
KTGT screening campaign. The amines consisted of primary,
secondary, aromatic and aliphatic fragments, covering a broad
range of chemical space and structural diversity, with many
compounds falling within the “Rule of 3” guidelines suggested
for FBDD (see the Supporting Information).8,9 All fragments
with known “toxicophores” were removed. By selecting a
diverse amine library, a robust synthetic methodology
compatible with a wide range of fragments could be
investigated for KTGT libraries as well as for the development
of covalent inhibitors.
binding site irreversibly captures an acrylamide-modified
fragment in a protein templated, conjugate addition reaction.
KTGT is able to identify fragments with low affinity for the
protein of interest, which has the potential to provide accurate
and relevant hit matter, enabling the development of fragments
into inhibitors or antagonists of challenging targets, such as
protein−protein interactions (PPIs).
Acrylamides have also been successfully used as electrophilic
capture groups in the development of covalent inhibitors,5 as
exemplified by the EGFR/ERBB2 inhibitor neratinib, which is
currently in phase III clinical trials for the treatment of
metastatic breast cancer, Figure 1.5c
In order to add value to KTGT as a fragment screening
technique, an efficient and high-yielding procedure for the
preparation of structurally relevant acrylamide libraries from
their corresponding amines was sought. Solution-phase
To prepare the acrylamides for screening, the 192-member
amine library was initially subjected to standard acylation
conditions using acryloyl chloride and triethylamine base using
an automated liquid-handling system.10 Unfortunately, the
success rate (determined as desired product isolated from the
reaction) was only 33%, and the average isolated product yield
Figure 1. Structure of neratinib, a covalent inhibitor of EGFR/ERBB2,
currently in phase III clinical trials.
Received: December 3, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/ol503486t
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Organic Letters
Letter
was a disappointing 24%, Table 1. The low success rate and
product yield were attributed to the susceptibility of
HPLC. Therefore, the improved Amberlyst A26 resin method-
ology provided access to acrylamide fragments that would
otherwise be unobtainable using the standard conditions, thus
enabling the inclusion of greater chemical diversity in our
acrylamide library.
Table 1. Comparison of Standard Acylation versus Our
Conditions for Acrylamide Synthesis
To exemplify the utility of the method, selected examples
from our library are outlined in Table 2. Entries 1−3 highlight
Table 2. Selected Examples Prepared Using the Amberlyst
A26 Method15
d
no. of
amines
total
successful
success rate average yield
c
method
(%)
(%)
standard
acylation
192
64
33
24
a
Amberlyst
30
25
83
54
b
resin
a
Reagents and conditions: acryloyl chloride (0.3 mmol), amine (0.3
b
mmol), NEt3 (0.9 mmol), CH2Cl2 (1 mL). Reagents and conditions:
acryloyl chloride (0.3 mmol), amine (0.3 mmol), Amberlyst 26 (OH
c
form) (45 mg), CH2Cl2/CHCl3 (1 mL). Success determined as
d
desired product isolated. Isolated, purified yield.
acrylamides to polymerize, as well as the need for extensive
purification to remove side products formed during the
reaction, due to the reaction of acryloyl chloride with
triethylamine.
We therefore chose to investigate alternative reaction
conditions that would enable acrylamide isolation without the
need for an aqueous workup or purification and in sufficient
purity for use in KTGT. Methods investigated included solid-
phase syntheses,6,7 boron-mediated amidations,11,12 solid-
supported amide coupling agents,13 as well as Lewis acid
mediated transformations.14 Unfortunately, these methods did
not overcome the limitations of low conversion, side product
formation, and polymerization. Attempts to mask the
acrylamide functionality, in order to prevent any residual
amine from reacting with the product acrylamide prior to
purification, did not deliver an efficient and high-throughput
method.
In order to prevent the unwanted side reactions under
standard acylation conditions between acryloyl chloride and the
amine base (in this case NEt3), inorganic and solid-supported
bases were investigated. Inorganic bases proved to be inefficient
due to their poor solubility in organic solvents and the
hydrolysis of acryloyl chloride under aqueous conditions.
Gratifyingly, ion-exchange resin Amberlyst A26 (OH-form)
provided an efficient method of scavenging the HCl generated
during the reaction, without reacting with acryloyl chloride to
generate unwanted side products. An optimized method was
therefore developed.15 It was envisaged that this method would
enable the rapid generation of acrylamide libraries from amine
starting points suitable for KTGT, particularly as the method
was suitable for relatively small-scale reactions (0.1−0.3 mmol
amine).
In order to provide a robust and thorough test for the
Amberlyst A26 method, a sample of 30 fragment amines were
selected from the initial 192 member library. This selection
was of amines that failed to react under standard acylation
conditions.10 They were subjected to the improved Amberlyst
A26 resin method.15 The 30-member test set once again
contained a range of primary, secondary, aromatic, and aliphatic
amines for diversity (see the Supporting Information). Of the
30 fragment amines, 25 reacted successfully, required no further
purification and gave a satisfactory average yield of 54%. All
examples were isolated in >95% purity as determined by
a
b
Isolated yields reported in ref 1. Average isolated yield when
prepared on a 0.3, 1.2, 5.0, and 10.0 mmol scale.
the improvement in yield when using our Amberlyst A26 resin
method compared with the standard acylation conditions. The
selection confirms that a range of functional groups are
tolerated and that primary and secondary amines react equally
well under these conditions. Often the presence of basic amine
centers can be problematic due to quaternization; however, N-
(1-benzylpiperidin-4-yl)acrylamide was prepared in 66% yield
B
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Organic Letters
Letter
(7) Sheng, S. R.; Wang, X. C.; Liu, X. L.; Song, C. S. Synth. Commun.
2003, 33, 2867−2872.
without the need for purification compared with the reported
literature yield of 31% (entry 3). In order to test whether this
methodology was applicable to the scale-up of acrylamide
fragments, cyclohexylamine was subjected to the Amberlyst
A26 resin method up to 10 mmol in comparable yields, Table 2
(entry 9).
Synthetic methods have been investigated for the rapid
conversion of amine libraries to acrylamides that are suitable for
use in KTGT screening campaigns. The reactive nature of the
acrylamide functional group poses challenges to the purification
and isolation of the desired fragment acrylamides. This was
overcome through the use of ion-exchange resin Amberlyst A26
(OH-form), which drives the reaction to completion without
the formation of unwanted side products. The short reaction
times and experimental simplicity of this method ideally lend it
for use in high-throughput combinatorial chemistry as well as
for the efficient synthesis of specific acrylamide-bearing
compounds that could be used as potential covalent inhibitors
against relevant therapeutic targets.
(8) Congreve, M.; Carr, R.; Murray, C. W.; Jhoti, H. A. Drug
Discovery Today 2003, 8, 876−877.
(9) Jhoti, H. A.; Williams, G.; Rees, D. C.; Murray, C. W. Nat. Rev.
Drug Discovery 2013, 12, 644−645.
(10) Standard acylation conditions: 192 amines (300 μmol) were
placed in 24 well trays, and the addition of Et3N (1 mL, 0.3 M in
CH2Cl2) was automated using parallel equipment. The vials were
shaken for 15 min. The vials were charged by hand with acryloyl
chloride (1 mL, 0.3 M in CH2Cl2). The vials were shaken for 16 h.
The samples were concentrated under reduced pressure, redissolved in
DMSO (1000 μL) and MeOH (200 μL), and then purified by
preparative HPLC. The fractions containing product were collected
and concentrated to dryness to give desired product.
(11) Starkov, P.; Sheppard, T. D. Org. Biomol. Chem. 2011, 9, 1320−
1323.
(12) Lanigan, R. M.; Starkov, P.; Sheppard, T. D. J. Org. Chem. 2013,
78, 4512−4523.
(13) Valeur, E.; Bradley, M. Tetrahedron 2007, 63, 8855−8871.
(14) Yadav, V. K.; Babu, K. G. J. Org. Chem. 2004, 577−580.
(15) Amberlyst A26 resin method conditions: 30 amines (300 μmol)
were placed in 24 well trays and dissolved in 900 μL of CHCl3. To
each vial was added acryloyl chloride (100 μL, 3 mM in CH2Cl2), and
the vials were shaken for 5 min, whereupon a solid precipitate was
formed. Subsequently, Amberlyst A26 (OH-form) (80 mg) was added
to each vial, which was shaken for a further 5 min. Amberlyst A26 resin
was removed in parallel using vacuum filtration and subsequently
washed with CH2Cl2 (5 mL), and the filtrate was concentrated under
reduced pressure to provide the desired product in >95% purity.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, general methods, and spectroscopic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Email: jeffrey.stonehouse@novartis.com.
Present Address
Global Discovery Chemistry, Novartis Institutes for Biomedical
Research, 250 Massachusetts Ave, Cambridge, MA 02139.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank David Pearce (Global Discovery Chemistry, Novartis
Horsham Research Centre, U.K.) for his assistance with the
combinatorial synthesis and purification. A.A. and C.E.A. are
grateful for Wellcome ISSF Networks of Excellence funding for
initial studies in this area.
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DOI: 10.1021/ol503486t
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