Multicomponent mapping of boron chemotypes furnishes selective enzyme inhibitors

Article abstract of DOI:10.1038/s41467-017-01319-4

Heteroatom-rich organoboron compounds have attracted attention as modulators of enzyme function. Driven by the unmet need to develop chemoselective access to boron chemotypes, we report herein the synthesis of α-and β-aminocyano(MIDA)boronates from borylated carbonyl compounds. Activity-based protein profiling of the resulting β-aminoboronic acids furnishes selective and cell-active inhibitors of the (ox)lipid-metabolizing enzyme α/β-hydrolase domain 3 (ABHD3). The most potent compound displays nanomolar in vitro and in situ IC₅₀ values and fully inhibits ABHD3 activity in human cells with no detectable cross-reactivity against other serine hydrolases. These findings demonstrate that synthetic methods that enhance the heteroatom diversity of boron-containing molecules within a limited set of scaffolds accelerate the discovery of chemical probes of human enzymes.

Full text of DOI:10.1038/s41467-017-01319-4

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DOI: 10.1038/s41467-017-01319-4
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1 Davenport Research Laboratories, Department of Chemistry, University of Toronto, 80 St George Street, Toronto, ON, Canada M5S 3H6. 2 Department of
Molecular Medicine, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550N. Torrey Pines Rd., La Jolla, CA 92037, USA. Joanne
Tan and Armand B. Cognetta III contributed equally to this work. Correspondence and requests for materials should be addressed to
A.K.Y. (email: ayudin@chem.utoronto.ca)
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oron-containing molecules (BCMs) have found widespread
utility as mechanistic probes and therapeutic agents1–6. The
breakthrough discoveries of proteasome inhibitors borte-
b
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R2
O
H
N
B
...
zomib7, ixazomib8, and delanzomib9 opened doors for synthetic
and medicinal chemists to explore the potential of boron in
therapeutic intervention. Akin to all molecules designed to
interact with protein targets, the structures of bioactive BCMs
must contain a sizable proportion of heteroatoms, including
nitrogen, oxygen, halogens, and sulfur. In the past, the absence of
heteroatoms in BCMs has resulted in lack of selectivity amongst
related families of enzymes10. BCMs display a reversible-covalent
mode of inhibition with serine proteases5. Boron has a unique
ability to adopt a range of coordination modes upon interaction
with protein targets11. This stands in contrast to other electro-
philes such as epoxides, aziridines, and Michael acceptors, which
...
N
H
R1
O
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a
Amino acids
R1
Aminoboronic acids
R1
(HO)2B
(HO)2B
R2
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α
α
NH
N
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O
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N
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α
α
R
B
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display
a singular type of interaction with active site
O
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OH R2
nucleophiles12–14. Despite the versatility and recent successes of
BCM-driven medicinal chemistry, there are still few examples of
boron-containing therapeutic agents. This can be partially
explained by the fact that synthetic technologies to site-selectively
introduce boron into heteroatom-rich environments remain
underdeveloped. The thermodynamic preference of boron to
migrate from carbon to oxygen or nitrogen, further aggravated by
the low kinetic barrier for these transformations15,16, accounts for
the dearth of available methods.
R2
CN
R1
(HO)2B
(HO)2B
NH
CN
NH
R2
R1
X = O, S, N
The structures of the most celebrated boron-containing che-
motherapeutics currently on the market are based on the α-
aminoboronic acid motif. Inspired by the impact of β-amino
acids and β-peptides on contemporary science17,18, we questioned
the potential significance of homology in aminoboronic scaffolds
and turned to amphoteric boron-containing compounds as the
enabling building blocks. The goal of our work was to build upon
facile α-aminoboronic acid synthesis19,20 and the recently
demonstrated stability of β-aminoboronic acids21.
Fig. 1
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of α-boryl aldehydes 1a–g with primary amines led to borylated
iminium ions 2a–g (Fig. 2a). Under reductive conditions, we were
able to access β-amino(MIDA)boronates 3 containing the
BCH2CH2N connectivity (Supplementary Methods)21, which
have been shown to play important roles in catalysis33. Building
on this observation, we were curious to evaluate the behavior of
borylated iminium ions in multicomponent reactions. The cya-
nide anion was chosen as the nucleophilic carbon-based com-
ponent due to its minuscule steric demand. Upon addition to
borylated iminium 2a, we were excited to see evidence of β-
Herein, we set out to prepare molecular frameworks containing
boron and heteroatoms of biological significance in order to map
the vicinity of the electrophilic boron warhead. For the design of
small heteroatom-rich BCMs, we wanted to ensure that the
parent scaffolds featuring mainly hydrogens off the connecting
chain could be perturbed by the smallest possible carbon sub-
stituent capable of productive interactions with proteins. The
chemically robust nitrile functionality came to our attention.
Nitriles are not readily metabolized22–26 and feature a short triple
bond. The rod-like nitrile geometry provides a carbon-based
substituent with a minuscule steric demand: based on A-values,
the CN unit is eight times smaller than the methyl group27. This
enables nitriles to project into narrow clefts in proteins and
engage in productive polar interactions and/or hydrogen bonds in
sterically challenging environments28. To append nitrile groups
to the chain connecting boron and nitrogen, we employ borylated
iminium ions of the recently developed α-boryl aldehydes29–31
and acylboronates32. The synthetic utility of borylated iminium
ions is derived from the N-methyliminodiacetic acid (MIDA)
substituent on boron, which mitigates boron’s propensity to
undergo C–to–O and C–to–N migrations and enables cellular
permeability. Empowered with this strategy, we have identified
selective and cell-active inhibitors of the (ox)lipid-metabolizing
enzyme α/β-hydrolase domain 3 (ABHD3).
aminocyano(MIDA)boronate
4
formation containing the
BCH2CH(CN)N motif (Fig. 2b). During the reaction optimiza-
tion, we initially hypothesized that benzoyl and acetyl cyanide
could act as nucleophilic sources of cyanide34. However, the
hydrolysis of the iminium ion was faster than the cyanide addi-
tion. We also tried using acetone cyanohydrin with catalytic
triethylamine35 and observed desired compound 4 as the major
product, but with poor conversion. We then identified tri-
methylsilyl cyanide as the most suitable reagent when used in the
presence of a Lewis acid35. Lowering the reaction temperature
resulted in poor solubility of α-boryl aldehyde 1a. The use of 3Å
molecular sieves to remove water had no influence on the reac-
tion. The reaction proceeded similarly in both acetonitrile and
dichloromethane. We chose to continue our studies with acet-
onitrile because it improved the solubility of α-boryl aldehyde 1a.
To be useful as chemical probes, β-aminocyano(MIDA)
boronates need to be stable in the free boronic acid form. We
anticipated that the nucleophilicity of the secondary nitrogen’s
lone pair might affect this stability. Indeed, this was the case when
we attempted to deprotect MIDA under various mildly acidic and
basic conditions. Therefore, we decided to attenuate the
nucleophilicity of the nitrogen’s lone pair by trapping it as an
amide. The β-aminocyano(MIDA)boronates were successfully
acylated using acid chlorides and highly electrophilic anhydrides
Results
Synthesis of the MIDA boronate library. We began our inves-
tigation with a model study shown in Fig. 1. Our recent studies of
borylated iminium ions21 opens doors to run a wide range of
multicomponent reactions with potential to expand the accessi-
bility of heteroatom-rich organoboron compounds. Condensation
2
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[B]
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such as trifluoroacetic anhydride. Compound 4b did not react dependent manner against HEK 293T lysates recombinantly
due to its poor nucleophilicity. We were also able to synthesize expressing ABHD3 confirmed boronate inhibition of ABHD3 and
the β-aminocyano(MIDA)boronates and acylate them in one pot identified 4j as the most potent and selective ABHD3 inhibitor
to provide β-aminocyano(MIDA)boronates 4d–l in higher yields (Supplementary Fig. 2), displaying an in vitro IC50 value of 0.14
compared to the two-step procedure (81% vs. 55%, respectively µM [95% confidence interval (CI, 0.097–0.20 µM)] (Fig. 5a, c).
for compound 4d). We also extended this chemistry to Compound 4j also inhibited ABHD3 in situ with a similar IC50
acylboronate 5, to provide α-aminocyano(MIDA)boronate 7, value [0.040 µM (CI, 0.026–0.060 µM)] (Fig. 5b, c). We also
the B,N-framework that is one carbon chain shorter (Fig. 2c). examined the relationship between FP-Rh incubation time and
To further expand the heteroatom mapping set, we prepared serine hydrolase engagement for 4j and related compounds, and
the BCO, BCS and BCN motifs (compounds 8–10) through found that the inhibition profiles were unchanged even at very
α-functionalization of alkyl(MIDA)boronates (Supplementary short time points (5 min), indicating that we did not overlook
Methods)20. A summary of each reaction scope is shown in Fig. 3. transient reversible inhibition events in our original 30 min assay
(Supplementary Fig. 3). Compound 4j was evaluated as the
racemate. It is likely that further improvements in potency will be
uncovered upon evaluation of the enantiomerically pure variants
MIDA boronate screening for serine hydrolase inhibition.
With the β-amino(MIDA)boronates and α- and β-aminocyano
of 4j. Studies aimed at the synthesis of enantiomerically pure β-
(MIDA)boronates in hand, we proceeded to test them for inhi-
aminoboronic acids are currently underway.
bitory activity against serine hydrolases (SHs). SHs represent a
large (> 200 member) enzyme family in mammals that plays
Structure-activity relationship analysis of ABHD3 hits. All five
important roles in metabolizing bioactive proteins, peptides, and
metabolites36. BCMs are able to interact with the conserved serine
of the ABHD3 hits contain a phenyl amide, which denotes its
importance for the potency. Replacing the phenyl amide with a
nucleophile of SHs through covalent reversible binding, rendering
the enzyme inactive5. We assessed boronate compounds against a
methyl amide in compounds 4d–f or the trifluoromethyl amide in
4l resulted in no notable ABHD3 inhibition. Compound 4j
appears equipotent with compounds 4h and 4g against ABHD3,
but is more selective against ABHD10. This increased selectivity
broad set of mammalian SHs by competitive activity-based pro-
tein profiling (ABPP) in mouse brain proteome37. All library
members were initially tested at 20 µM (30 min pre-incubation),
and SH inhibition evaluated using the broad-spectrum activity-
based probe fluorophosphonate-rhodamine (FP-Rh), where
reductions in FP-Rh labeling of one or more SHs in the brain
observed for 4j may be a result of the highly electronegative
fluorine atom, which is known to have profound pharmacological
effects when installed in key positions39.
proteome was detected by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and in-gel fluorescence scanning. Boronate 4j is a selective ABHD3 inhibitor. ABHD3 is an
Multiple members of the boronate library were found to inhibit a integral membrane enzyme that is enriched in brain tissue, where
low-abundance, ~40 kDa SH previously identified in previous it hydrolyzes medium chain and oxidatively modified phospho-
ABPP studies as ABHD338 (Fig. 4, Supplementary Fig. 1). Follow- lipids40. Few inhibitors have been reported for ABHD3 and these
up ABPP of hit compounds screened in a concentration- compounds lack selectivity across the SH class38. Our initial gel-
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Objective
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based ABPP experiments suggested that 4j exhibited good human colon cancer cell line SW620 prepared for quantitative
selectivity for ABHD3 (Fig. 4, Supplementary Fig. 2), but only a MS-based ABPP using stable isotope labeling with amino acids in
limited number of SHs (~20) can be resolved by SDS-PAGE cell culture (SILAC)[42]. At both tested concentrations, 4j fully
analysis of tissue proteomes. We therefore next evaluated 4j using blocked (95%+) ABHD3 activity without exhibiting any cross
higher-resolution, mass spectrometry (MS)-based ABPP meth- reactivity with over 60 additional SHs quantified in SW620 cells
ods41. We tested 4j at two concentrations –0.5 and 10 µM – in the (Fig. 5d). Notably, 4j maintained high specificity for ABHD3 over
4
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