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V. Nemec, L. Maier, B.-T. Berger et al.
European Journal of Medicinal Chemistry 215 (2021) 113299
heterocycles as central pharmacophores [15]. The furo [3,2-b]pyr-
idine motif belongs to the relatively underexplored ones; however,
we recently discovered that it can be used as the central scaffold of
potent and highly selective kinase inhibitors [16] and MU1210 has
been recently recommended as a quality chemical biology probe for
cdc-like kinase 1/2/4 (CLK1/2/4) [17]. The biology and therapeutic
potential of cdc-like kinases (CLKs) have been explored mainly over
the last decade [17] and the first CLK inhibitor, compound
SM08502, entered clinical trials only recently [18].
Many CLK inhibitors, including MU1210, show off-target activity
against structurally closely related homeodomain-interacting pro-
tein kinases (HIPKs) and dual-specificity tyrosine phosphorylation-
regulated kinases (DYRKs) [17]. Identification of inhibitors selec-
tively targeting only one of those sub-families is a non-trivial task
since these kinases are structurally very closely related [19].
HIPKs have been associated with diverse signaling roles
including DNA damage response [20], regulation of expression of
microRNAs [21] and autophagy [22] suggesting implication of this
kinases in the development of diverse diseases. Nevertheless, HIPKs
still belong to relatively underexplored kinases, which could be in
part attributed to the lack of quality chemical biology probes.
Generally, small-molecule HIPK inhibitors are quite rare and
none of the compounds published to day fulfil the criteria for a
quality chemical probe. Of the reported compounds, TBID exhibits
only modest activity in vitro (HIPK2 IC50 ¼ 330 nM) and low celullar
activity, and its selectivity profile was determined via screening
modification of the sequence we used previously for its silylated
analogue 8 (route B in Scheme 1) [16]. Gratifyingly, and in contrast
to the bromo-analogue 1, with compound 9 we were able to
execute the desired chemoselective couplings, first at position 3
and then at position 5. Since compound 9 can be prepared easily on
gram scale, we used it in second-generation synthesis of the CLK
probe MU1210 (route B in Scheme 1). Both the number of steps (6
vs. 9 from commercially available 6-chloropyridin-3-ol 2) and the
overall yield (39% vs. 23%) in the new route B are significantly
improved compared to the original route A [16].
We also prepared new analogues of MU1210, expanding the
existing SAR [16] (Table 1). The partially saturated analogues 12a
and 12b (prepared from 5 by the previously reported methodology)
[16] showed weaker inhibition of CLKs than MU1210. However, the
potency and selectivity of the pyrazole-containing analogues 12c,
12d, and 12e (prepared by the new route from 9) were comparable.
In order to assess their activity in the cell, the compounds 12c
and 12d were profiled along with MU1210 in the MCF-7 cell line
(Fig.1), which we used previously to quantify the cell-based activity
of furo [3,2-b]pyridine-based CLK inhibitors [16] to assess their
therapeutic potential in this context [28].
In comparison to MU1210, 12c showed weaker and 12d signif-
icantly weaker activity.
The compounds 12c and 12d were also profiled in vivo e we
determined their pharmacokinetic profile in the mouse under the
conditions that were directly comparable to those used for MU1210
(i.e. IV and IP dosing, 10 mpk of the dihydrochloride salt) [16]. As
illustrated by Fig. 2, the pharmacokinetic profiles of both com-
pounds are inferior to that of the CLK chemical biology probe
MU1210.
against 76 kinases at (in our opinion insufficiently high) 1
mM
concentration [23]. The more potent compound CVM-6-139-1
(HIPK2 IC50 ¼ 74 nM) has moderate selectivity profile across the
kinome (determined via screening against 353 kinases at 10 mM
concentration) [24]. The allosteric HIPK2 inhibitor BT173 has been
claimed to be selective; however, the data on its selectivity or target
engagement are not available [25]. Search in the Probe Miner
compounds (e.g. ruboxistaurin, PHA-665752, foretinib or sorafenib)
with decent in vitro activity (IC50 ˂ 100 nM) against HIPK1-4 but
rather poor selectivity. Several classes of reported CLK inhibitors
also show off-target inhibitory activity against HIPKs [17].
In this study, we report new findings in the area of synthesis and
biological profiling of substituted furo [3,2-b]pyridines. Specifically,
newly developed synthetic route allowed for efficient second-
generation synthesis of the CLK chemical probe MU1210, prepara-
tion of its new analogues, and identification of new highly selective
inhibitors of HIPKs.
Collectively, both cell-based and in vivo data sets further illus-
trate the fact that of the numerous furo [3,2-b]pyridines prepared
thus far, the compound MU1210 possesses best overall profile, and
confirm its selection as quality chemical biology probe for CLKs.
Despite not identifying a more active CLK compound, the pro-
files of MU1210, 12d and 12e, along with some additional previ-
ously profiled compounds containing unsubstituted pyrazole at
position 5 of the furo [3,2-b]pyridine scaffold [16], suggested that
the scaffold could also deliver potent HIPK inhibitors. The HIPK
family of proteins plays diverse, and sometimes seemingly con-
flicting, biological roles in normal development and disease; their
function is regulated by many ways and recent genetic studies
revealed their pleiotropic and essential physiological roles [29,30].
Given the diverse signaling pathways regulated by HIPKs, it is likely
that they act to fine-tune signaling and may mediate cross talk in
certain contexts, and their context-specific activities play role in
diseases such as cancer and fibrosis [29,31]. Correspondingly,
recent findings indicate that inhibition of HIPKs could be an
attractive strategy for the treatment certain malignancies [32e34].
Bona fide HIPK inhibitors could be used as chemical biology probes
for unraveling the complex HIPK biology; therefore, given that such
molecules are quite rare [23e25] and taking into account the
profiles of MU1210, 12d and 12e, we considered re-purposing the
furo [3,2-b]pyridine scaffold towards selective HIPK inhibitors as
justified and promising endeavor.
2. Results and discussion
The original synthesis of MU1210 (and its analogues) relied on
sequential chemoselective couplings of proper 3,5-dihalogenated
furo [3,2-b]pyridine precursor. While the differences in reactivity
of the halogens in previously known 3-bromo-5-chlorofuro [3,2-b]
pyridine (1) was not sufficient, the route utilizing newly prepared
3-bromo-5-iodofuro [3,2-b]pyridine (5) was successful, ultimately
It should be noted that while compound 5 can be prepared on
gram scale, its synthesis is technically not trivial e the Finkelstein
reaction used to install iodine at position 5 requires long reaction
times and precise continuous addition of acetyl chloride in large
excess over the reaction time [16]. Palladium-catalyzed couplings
with 5 proceed first at position 5 - the resulting products are thus
optimal for late-stage manipulation of position 3.
In order to explore new compounds as potential HIPK inhibitors,
we utilized the intermediate 9 and systematically varied the posi-
tions 3 and 5 thereby preparing set of the target compounds 21e-k
butylphenylboronic acid proceeds practically quantitatively and
with exclusive chemoselectivity e in selected cases this step was
thus combined with the following coupling within one-pot pro-
cedure that directly afforded the target compounds (21e and 21k,
see SI). When we attempted the second coupling (in order to install
the pyrazole motif) with pyrazole pinacolboronate, the reaction
In the course of the project, we wished to explore the reversed
sequence e i.e. initial manipulation of position 3 followed by final
functionalization of position 5. Along that line, we prepared here-
tofore unknown 3-iodo-5-chlorofuro [3,2-b]pyridine (9) by
2
Němec, Václav
