Late-stage synthesis and application of photoreactive probes derived from direct benzoylation of heteroaromatic C-H bonds

Article abstract of DOI:10.1039/d0ob00336k

A C-H functionalization strategy for the expedient access to photoreactive chemical probes of commonly found heterocyclic fragments or drug molecules of pharmaceutical relevance is described. A series of aryl glyoxylic acid reagents featuring pendant alkyne or azide clickable handles have been developed for application in the radical-mediated appendage of benzoyl fragments onto simple heteroaromatic fragments, as well as more complex drug-like compounds. This unprecedented strategy of chemical probe synthesis allows for direct access to photoreactive chemical probes without any requirement of fragment pre-functionalization or significant synthetic re-evaluation.

Full text of DOI:10.1039/d0ob00336k

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Late-stage synthesis and application of
photoreactive probes derived from direct
Cite this: DOI: 10.1039/d0ob00336k
benzoylation of heteroaromatic C–H bonds†
Received 14th February 2020,
Accepted 23rd April 2020
Kevin D. Hesp, * Jun Xiao and Graham M. West
DOI: 10.1039/d0ob00336k
rsc.li/obc
A C–H functionalization strategy for the expedient access to the additional synthetic evaluation required to introduce suit-
photoreactive chemical probes of commonly found heterocyclic able synthetic handles onto target ligands to facilitate incor-
fragments or drug molecules of pharmaceutical relevance is poration of benzophenone without disrupting key binding
described.
A series of aryl glyoxylic acid reagents featuring features can present challenges for photoreactive probe
pendant alkyne or azide clickable handles have been developed for design. In addition to the application of minimalist photo-
3
application in the radical-mediated appendage of benzoyl frag- reactive probe designs (Fig. 1A), the advent of fully-functio-
ments onto simple heteroaromatic fragments, as well as more nalized fragment probes, which imbed the photoreactive
complex drug-like compounds. This unprecedented strategy of group into the ligand of interest, have presented an opportu-
chemical probe synthesis allows for direct access to photoreactive
chemical probes without any requirement of fragment pre-
functionalization or significant synthetic re-evaluation.
Introduction
Benzophenone-derived photoaffinity probes have a longstand-
ing history for interrogating specific ligand–protein inter-
actions along complex biochemical pathways in the field of
1
chemical biology. In particular, such probes have been routi-
nely leveraged for the identification of new targets for known
biologically active compounds, as well as for elucidating the
binding site or mechanistic subtleties of a specific ligand
within a known target. The popularity of benzophenone is in
part related to its high chemical stability and ready availability
1
b,-
when compared to related azide and diazirine photophores,
c,2
but is also heavily rooted in its unique ability to reversibly
generate reactive radical intermediates without probe destruc-
tion at non-protein damaging wavelengths.
Despite these advantages, the inherent lipophilicity and
steric profile of the benzophenone moiety persist as limit-
ations from the perspective of increased non-specific protein
binding, as well as sterically-driven binding differences when
compared to unmodified parent ligands or fragments. Often Fig. 1 (A) Current strategy for benzophenone photoreactive probe syn-
thesis from pre-functionalized fragments; (B) proposed investigation of
streamlined probe synthesis by late-stage C–H functionalization on
Pfizer, Inc., Medicine Design, Eastern Point Road, Groton, Connecticut, 06340, USA.
E-mail: kevin.hesp@pfizer.com
unmodified parent ligands or fragments; (C) use of arylglyoxylic acid
reagents with pendant alkynes for the synthesis of heterocyclic benzo-
phenone probes via radical benzoylation of heteroaromatics in biologi-
cally active molecules.
†
Electronic supplementary information (ESI) available: Experimental details and
supporting characterization. See DOI: 10.1039/d0ob00336k
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nity to alleviate aspects of these limitations in the context
4
integrated phenotypic screening and target identification. In
ð1Þ
order to expand the synthetic accessibility and structural
diversity of this concept, we became interested in the identifi-
cation of synthetic options for the late stage benzoylation of
unmodified fragments and parent ligands (Fig. 1B). Ideally
this strategy would rely solely on the innate reactivity of com-
monly encountered heterocycles, which are pervasive in frag-
ments and bioactive molecules of pharmaceutical interest. If
successful, this strategy could have significant impact on the
ability to rapidly access photoreactive probe fragment
libraries with minimal synthetic investment from large sub-
strate diversity to expedite new biological target identification
efforts.
Following the identification of 1b as an optimal reagent,
the scope of heteroaromatic benzoylation was initiated with a
focus on defining the reactivity and regioselectivity trends on
simple heteroarene fragments to guide its use for more elabor-
ate photoreactive probe synthesis on complex drug-like struc-
8
tures (Scheme 1). In general, most classes of heterocycles
were reactive using the standard conditions, but those that
were either inherently electron-deficient or featured electron-
withdrawing substituents typically proceeded with superior
conversions to the C–H benzoylation products. Indeed, simple
pyridine derivatives featuring electron-withdrawing groups in
the 4-position (2c–2e) were readily benzoylated at the 2-posi-
tion to give the corresponding 2-benzoyl pyridine products in
good yields. Whereas benzoylation of methyl 3-methyl picoli-
nate (2f) gave a mixture of regioisomers slightly favouring the
C6-substituted product (3f : 3f′ = 1.5 : 1), the use 3-methoxypyri-
dine selectively reacted at the 2-position to give 3g in 27% iso-
lated yield. The methodology was successfully extended to
include substituted diazines that are commonly found in drug-
like molecules, such as pyridazine (2h–2j), pyrazine (2k), and
pyrimidine (2l), with good yields and high regioselectivity in
most examples. In addition to monocyclic heterocycles, the
use of [6,6]- and [5,6]-ring systems, such as quinoline (2m), iso-
quinoline (2n), imidazopyrimidine (2o), and pyrazolopyrimi-
dine (2p), were also readily benzoylated providing access to the
The use of radical chemistry has been established as a
practical and selective strategy for the functionalization of
5
complex molecules in a predictable manner. The most
appealing attributes of radical-based chemistry in the context
of photoreactive probe synthesis are the opportunities to alle-
viate heightened synthetic costs and time for preparing
complex molecules featuring minor modifications to the
parent compounds, as well as the innate tolerance of mole-
cular complexity and reactive functional groups. Given these
clear benefits, we focused our efforts on the identification of
suitable benzoyl radical precursors for the development of an
efficient late-stage synthesis of benzophenone-like photoreac-
tive probes.
Based on these considerations, we report the identification
and reactivity of readily accessible alkyne-substituted, aryl
glyoxylic acid reagents for application in the mild C–H ben-
zoylation of both simple heteroarene fragments, as well as
complex biologically active molecules, to provide streamlined
access to benzophenone-like photoreactive probes (Fig. 1C).
Of importance, this strategy offers potential advantages over
more classical benzophenone in the context of developing
ligand-efficient and less lipophilic photoreactive chemical
probes that are rapidly accessible with minimal synthetic
assessment.
Results and discussion
6
Building on the chemistry pioneered by Minisci, and in line
with the goals of this study, we prepared a series of substituted
aryl glyoxylic acids that featured clickable alkyne or azide func-
7
tional handles. Optimization of the aryl glyoxylic acid substi-
tution led to the identification of propargyl ether 1b, which
provided the 2-benzoyl pyrazine product 3b in a comparable
yield to that observed in our initial test reaction employing
6
a
unsubstituted phenyl glyoxylic acid 1a (eqn (1)). Of impor-
tance, these initial results established that the alkyne clickable
handle is unreactive under the radical forming conditions
and, thus, provided the impetus for continued pursuit of a het-
eroaromatic C–H benzoylation strategy for photoreactive probe
synthesis.
Scheme 1 Substrate scope for benzoylation of heteroaromatic frag-
ments using aryl glyoxylic acid 1b. The corresponding bis addition
product was isolated in a 18% yield.
a
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benzophenone-like compounds 3m–3p. Notably, several sub- recent campaign in search of a novel PDE9A inhibitor for treat-
1
1
strates that featured multiple potential reaction sites exhibited ment of cognitive disorders, we sought to showcase and vali-
complete regioselectivity, which resulted in only a single obser- date this strategy for the expedient identification of a photo-
vable regioisomer with C–H benzoylation proceeding at elec- reactive chemical probe of the clinical candidate 2q for PDE9A
tron-deficient positions.
binding site interrogation.
Building on the observed reactivity trends presented in
In addition to the facile introduction of the photoreactive
Scheme 1, we evaluated the methodology using more complex group in a single step from unmodified parent ligand 2q, the
chemical structures that would be more commonly encoun- resulting probe 3q, as well as the direct benzophenone com-
tered in drug discovery programs. Using our standard con- parator 4, were observed to maintain comparable potency
ditions, a range of biologically active compounds were readily against PDE9A (Fig. 3). In line with the overarching goals of
functionalized using the C–H benzoylation protocol to provide this study, the resulting minimalist probe 3q favourably offset
rapid access to benzophenone-like photoreactive probes the overall probe lipophilicity when compared to the benzo-
(
Fig. 2). In line with the reactivity trends identified in phenone derived probe 4, as judged by cLogP and the moder-
1
2
Scheme 1, the pyrimidine-containing PDE9A inhibitor, seroto- ate impact on the lipophilic efficiency (LipE) (Fig. 3).
nin 5-HT1A receptor partial agonist, and BACE inhibitor were Moderation of this key physicochemical property presents
selectively functionalized to provide access to the benzophe- opportunities for the design of chemical probes with favour-
none-like products in 12% (3q), 9% (3r), and 11% (3s) yields, able cell permeability and the potential mitigation of unpro-
9
respectively. Additionally, an mGluR5 modulator and the ductive non-specific protein binding – two important factors
9
smoking cessation drug Chantix, which feature highly substi- when considering their use in cell-based phenotypic screening
tuted pyrazine and quinoxaline cores, were readily benzoylated or photoaffinity studies. The measured reduction of lipophilic
in modest yields when employing aryl glyoxylic acid 1b under character between probes 3q and 4 was also manifest following
the standard radical forming conditions (3t; 39% and 3u; 32%, investigation into the off-target pharmacology within the PDE
respectively). Notably, the C–H functionalization chemistry protein family. Indeed, the more lipophilic probe 4 had a
proceeds in the presence of potentially reactive functional marked increase in potency against related PDE proteins,
7
,13
groups, such as the pyrimidinone and thioamidine groups or while 3q maintained selectivity for PDE9A (Fig. 3).
basic secondary amines. Although low to moderate isolated In light of the conserved potency of 3q and 4 against
yields are often observed, the most powerful feature of this PDE9A, as well as the proximity of the binding pocket to a
1
1,14
methodology is the ability to rapidly access photoreactive potentially reactive methionine residue,
we reasoned that
probes for assessment in key chemical biology applications photoactivation of 3q and 4 in the presence of PDE9A were
without the requirement of additional synthetic assessment or ideal experiments to test the relative propensity of a hetero-
functional group installation.
cyclic benzophenone to participate as a photo-crosslinking
There are few examples of heterocyclic benzoyl derivatives functional group. Incubation of recombinant PDE9A protein
1
0
behaving as ketyl radical precursors and scarce precedent for with a 10-fold excess of 3q under photoirradiation using
their use in interrogating protein–ligand interactions via a 365 nm UV-light for 30 minutes, followed by deconvulated
4
b
photo-initiated crosslinking event. In this regard, it was criti- mass spectroscopic analysis, revealed that
a successful
cal to exemplify the use of a representative ligand-benzoyl covalent modification of the protein had occurred with 34%
structural motif as a competent source of a ketyl-like radical in photolabeling efficiency (M + 553 observed; based on relative
order to validate the synthetic strategy for more broad appli-
cation in the chemical biology community. In light of Pfizer’s
Fig. 2 Application of late-stage photoreactive probe synthesis to drug
molecules.
Fig. 3 Comparison of potency, properties, and selected off-target
pharmacology for 2q, 3q, and 4.
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ology, was shown to be effective in the key photoaffinity label-
ing of the PDE9A protein while maintaining favorable drug-
like physicochemical properties. Given the operational simpli-
city and broad scope of compatible heteroarenes, this powerful
strategy holds promise to complement more classical photo-
reactive probe identification and is poised to rapidly impact
the interrogation of protein–ligand interactions, among other
chemical biology applications.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors thank Vincent Mascitti, Christopher am Ende,
Whitney Nolte, and Brian Raymer for helpful discussions,
Steven Jenkinson for potency determination of 3q and 4,
Ingrid Stock for assistance in acquiring PDE9A protein, Lise
Hoth and Thomas McLellan was mass spectrometry assist-
ance, and Matthew Teague and Thomas O’Connell for
HRMS acquisition. All noted individuals are Pfizer
colleagues.
Fig. 4 PDE9A photolabeling experiments with 3q and 4.
7
,15
ion intensities) (Fig. 4).
When the same experiment was
conducted in the presence of the probe 4, which featured the
more classical benzophenone photophore, a similar photola-
beling efficiency of 35% was observed (M + 551 observed;
7
based on relative ion intensities). The results of this direct
Notes and references
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variants of benzophenone represent comparable photoreactive
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See ESI† for additional details.
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9
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